Gene Therapy For Neurodegenerative Disorders

ABSTRACT

Compositions and methods for treating disorders affecting motor function, such as motor function affected by disease or injury to the brain and/or spinal cord, are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT/US2010/001239 filed Apr. 27,2010, which claims the benefit under 35 USC § 119(e)(1) of U.S.Provisional Application No. 61/174,982, filed May 2, 2009 and61/268,059, filed Jun. 8, 2009, which applications are incorporatedherein by reference in their entireties.

TECHNICAL FIELD

The present invention relates generally to gene delivery methods. Inparticular, the invention relates to compositions and methods fortreating disorders affecting motor function, such as motor functionaffected by disease or injury to the brain and/or spinal cord.

DESCRIPTION OF THE INVENTION

Gene therapy is an emerging treatment modality for disorders affectingthe central nervous system (CNS). CNS gene therapy has been facilitatedby the development of viral vectors capable of effectively infectingpost-mitotic neurons. The central nervous system is made up of thespinal cord and the brain. The spinal cord conducts sensory informationfrom the peripheral nervous system to the brain and conducts motorinformation from the brain to various effectors. For a review of viralvectors for gene delivery to the central nervous system, see Davidson etal., Nature Rev. (2003) 4:353-364.

Adeno-associated virus (AAV) vectors are considered useful for CNS genetherapy because they have a favorable toxicity and immunogenicityprofile, are able to transduce neuronal cells, and are able to mediatelong-term expression in the CNS (Kaplitt et al., Nat. Genet. (1994)1:148-154; Bartlett et al., Hum. Gene Ther. (1998) 9:1181-1186; andPassini et al., J. Neurosci. (2002) 22:6437-6446).

One useful property of AAV vectors lies in the ability of some AAVvectors to undergo retrograde and/or anterograde transport in neuronalcells. Neurons in one brain region are interconnected by axons to distalbrain regions thereby providing a transport system for vector delivery.For example, an AAV vector may be administered at or near the axonterminals of neurons. The neurons internalize the AAV vector andtransport it in a retrograde manner along the axon to the cell body.Similar properties of adenovirus, HSV, and pseudo-rabies virus have beenshown to deliver genes to distal structures within the brain (Soudas etal., FASEB J. (2001) 0.1:2283-2285; Breakefield et al., New Biol. (1991)2:203-218; and deFalco et al., Science (2001) 291:2608-2613).

Several experimenters have reported that the transduction of the brainby AAV serotype 2 (AAV2) is limited to the intracranial injection site(Kaplitt et al., Nat. Genet. (1994) 8:148-154; Passini et al., J.Neurosci. (2002) 22:6437-6446; and Chamberlin et al., Brain Res. (1998)22; 169-175). There is also evidence that retrograde axonal transport ofneurotrophic viral vectors, including AAV and lentiviral vectors, canalso occur in select circuits of the normal rat brain (Kaspar et al.,Mol. Ther. (2002) 5:50-56; Kasper et al., Science (2003) 301:839-842 andAzzouz et al., Nature (2004) 429:413-417. Roaul et al., Nat. Med. (2005)11(4):423-428 and Ralph et al., Nat. Med. (2005) 11(4):429-433 reportthat intramuscular injection of lentivirus expressing silencing humanCu/Zn superoxide dismutase (SOD) interfering RNA retarded disease onsetof amyotrophic lateral sclerosis (ALS) in a therapeutically relevantrodent model of ALS.

Cells transduced by AAV vectors may express a therapeutic transgeneproduct, such as an enzyme or a neurotrophic factor, to mediatebeneficial effects intracellularly. These cells may also secrete thetherapeutic transgene product, which may be subsequently taken up bydistal cells where it may mediate its beneficial effects. This processhas been described as cross-correction (Neufeld et al., Science (1970)169; 141-146).

A property of the recombinant AAV vectors described above is therequirement that the single-stranded DNA (ssDNA) AAV genome must beconverted into into double-stranded DNA (dsDNA) prior to expression ofthe encoded transgene. This step can be circumvented by the use ofself-complementary vectors which package an inverted repeat genome thatfolds into dsDNA without requiring DNA synthesis or base-pairing betweenmultiple vector genomes, thereby increasing efficiency of AAV-mediatedgene transfer. For a review of self-complementary AAV vectors, see e.g.,McCarty, D. M. Molec. Ther. (2008) 16:1648-1656.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromusculardisorder caused by mutations in the survival motor neuron 1 (SMN1) geneand loss of encoded SMN protein (Lefebvre et al., Cell (1995)80:155-165). The lack of SMN results in motor neuron degeneration in theventral (anterior) horn of the spinal cord, which leads to weakness ofthe proximal muscles responsible for crawling, walking, neck control andswallowing, and the involuntary muscles that control breathing andcoughing (Sumner C. J., NeuroRx (2006) 1:235-245). Consequently, SMApatients present with increased tendencies for pneumonia and otherpulmonary problems such as restrictive lung disease. The onset ofdisease and degree of severity are determined in part by the phenotypicmodifier gene SMN2, which is capable of making a small amount of SMN(Monani et al., Hum. Mol. Genet. (1999) 1:1177-1183; Lorson et al.,Proc. Natl. Acad. Sci. USA (1999) 96:6307-6311). Thus, patients with ahigh SMN2 copy number (3-4 copies) exhibit a less severe form of thedisease (referred to as Types II or III), whereas 1-2 copies of SMN2typically result in the more severe Type I disease (Campbell et al., Am.J. Hum. Genet. (1997) 61:40-50; Lefebvre et al., Nat. Genet. (1997)16:265-269). Currently, there are no effective therapies for SMA.

A fundamental strategy for treating this monogenic disorder is toincrease SMN levels in SMA patients. One approach to accomplish this isto modulate the endogenous SMN2 gene with small molecules that activatethe SMN2 promoter or correct the SMN2 pre-mRNA splicing pattern. Thealteration of SMN2 splicing also can be realized with antisenseoligonucleotides and trans-splicing RNAs. However, while modulating SMN2in vitro increased SMN levels and reconstituted nuclear gems in SMA celllines, efficacy studies with small molecule drugs have not translated tomeasurable improvements in the clinic (Oskoui et al., Nerotherapeutics(2008) 1:499-506).

SUMMARY OF THE INVENTION

The present invention is based on the discovery that both conventionalrecombinant AAV (rAAV) virions, as well as recombinantself-complementary AAV vectors (scAAV), are able to deliver genes to theCNS with successful expression in the CNS and treatment ofneurodegenerative disease. This therapy approach for the delivery ofgenes encoding therapeutic molecules that result in at least partialcorrection of neuropathologies provides a highly desirable method fortreating a variety of neurodegenerative disorders, including SMA.

Thus in one embodiment, the invention is directed to aself-complementary adeno-associated virus (scAAV) vector comprising apolynucleotide encoding a protein that modulates motor function in asubject with a motor neuron disorder. In certain embodiments, the motorneuron disorder is selected from spinal muscular atrophy (SMA),amytrophic lateral sclerosis (ALS), spinal bulbar muscular atrophy,spinal cerebellar ataxia, primary lateral sclerosis (PLS), or traumaticspinal cord injury.

In additional embodiments, the polynucleotide present in the scAAVvector encodes a survival motor neuron (SMN) protein. In certainembodiments, the SMN protein is human SMN-1. In further embodiments, theSMN-1 comprises an amino acid sequence with at least 90% sequenceidentity to the sequence depicted in FIG. 9B. In additional embodiments,the SMN-1 comprises an amino acid sequence as depicted in FIG. 9B.

In yet further embodiments, the invention is directed to a recombinantAAV virion, comprising an scAAV vector as described above.

In additional embodiments, the invention is directed to a compositioncomprising a recombinant AAV virion as above and a pharmaceuticallyacceptable excipient.

In further embodiments, the invention is directed to a method ofmodulating motor function in a subject with a motor neuron disordercomprising administering a therapeutically effective amount of thecomposition above to cells of the subject. In certain embodiments, thecomposition is administered to cells in vitro to transduce the cells andthe transduced cells are administered to the subject. In alternativeembodiments, the composition is administered to cells in vivo.

In further embodiments, the invention is directed to a method ofproviding an SMN protein to a subject with spinal muscular atrophy (SMA)comprising administering a recombinant AAV virion comprising an AAVvector as described above to cells of a subject in need thereof. Incertain embodiments the composition is administered to cells in vitro totransduce the cells and the transduced cells are administered to thesubject. In alternative embodiments, the composition is administered tocells in vivo.

In each of the methods above, the composition can be administered viadirect spinal cord injection. In other embodiments, the composition isadministered via intracerebroventricular injection. In additionalembodiments, the composition is administered into a cerebral lateralventricle. In certain embodiments, the composition is administered intoboth cerebral lateral ventricles. In other embodiments, the compositionis administered via both intracerebroventricular injection and directspinal cord injection. In additional embodiments, the composition isadministered by intrathecal injection.

These and other embodiments of the subject invention will readily occurto those of skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows survival of mice treated with AAVhSMN1 versus untreated SMAmice. Treatment with AAVhSMN1 increased survival in SMA mice. UntreatedSMA mice (n=34, open circles) had a median life span of 15 days. SMAmice treated at P0 with AAVhSMN1 (n=24, closed circles) had a medianlifespan of 50 days (p<0.0001), which was a +233% increase in longevity.

FIGS. 2A-2C show the effect of gene therapy treatment on SMN levels inthe spinal cord. Shown are hSMN protein levels in injected lumbar (FIG.2A), thoracic (FIG. 2B) and cervical (FIG. 2C) segments compared tountreated SMA and wild-type mice. Western blots were performed on thelumbar, thoracic and cervical segments of the spinal cord at 16, 58-66and 120-220 days after injection. The western blots from the threesegments were quantified and, to control for protein levels, SMN wasnormalized to β-tubulin and plotted as a percentage of age-matched wildtype. Key (and n-values): SMA, untreated knockout (n=5 at 16 days); AAV,AAV8-hSMN-treated SMA mice (n=7 at 16 days, n=5 at 58-66 days); scAAV,scAAV8-hSMN-treated SMA mice (n=5 at each time point). Values representthe mean±SEM.

FIGS. 3A-3J show the sub-cellular distribution of hSMN protein andexpression in motor neurons of the spinal cord in treated and untreatedSMA mice. hSMN protein was abundantly detected in the cytoplasm oftransduced cells (FIGS. 3A and 3B). Furthermore, hSMN protein wasdetected in the nucleus, as illustrated by the pair of gem-likestructures (arrowhead) magnified in the inset (FIG. 3A). hSMN proteinwas also detected in the dendrites (FIGS. 3B and 3C) and axons (FIG. 3D)of neurons. hSMN protein was not detectable on the tissue sections fromuntreated SMA mice (FIG. 3E). Co-localization of hSMN protein (FIG. 3F)with mouse ChAT (FIG. 3G) showed that a subset of transduced cells weremotor neurons (FIGS. 3H and 3I). The percentage of ChAT cells that wereimmuno-positive for hSMN protein was determined at 16 (white bars) and58-66 (black bars) days (FIG. 3J). Values represent the mean±SEM.

FIG. 4 shows motor neuron cell counts in the spinal cord in treated anduntreated SMA mice. Shown are the average numbers of ChATimmuno-positive neurons counted on 10 μm tissue sections for each group.Numbers represent counts of every tenth section from different levels ofthe cervical, thoracic, lumbar and sacral segments. Values represent themean SEM. Key: *, p<0.05; **, p<0.01; ***, p<0.001.

FIGS. 5A-5C show the myofiber cross-section area from muscle groups intreated and untreated SMA mice. The myofiber cross-section area frommultiple muscle groups was increased with AAVhSMN1 treatment. Stackedgraphs of the quadriceps, gastrocnemius and intercostal muscles from 16(FIG. 5A) and 58-66 (FIG. 5B) days showed that the distribution ofmyofiber sizes were similar between the treated SMA and the wild typemice. The overall average at 16 days showed that the myofibercross-section area was significantly higher with treatment (FIG. 5C).Furthermore at 58-66 days, the average area was statistically similarbetween treated SMA mice and age-matched wild-type in the gastrocnemiusand intercostal muscles (FIG. 5C). Values represent the mean±SEM. Key:WT, untreated wild type; HET, untreated heterozygote; SMA, untreatedknockout; AAV, AAVhSMN1-treated SMA mice; *, p<0.05; **, p<0.01; ***,p<0.001.

FIGS. 6A-6F show the structure of the NMJ in muscles in treated anduntreated SMA mice. The structure in the quadriceps, gastrocnemius, andintercostal was improved with gene therapy. Shown are the neuromuscularjunction (NMJ) from the quadriceps of untreated SMA (FIG. 6A), treatedSMA (FIG. 6B), and untreated wild type (FIG. 6C) mice at 16 days, andfrom treated SMA (FIG. 6D) and untreated wild type (FIG. 6E) mice at58-66 days. The pre- and post-synaptic NMJ was labeled with aneurofilament antibody (green) and with α-bungarotoxin staining (red),respectively. The arrowhead in the main panel points to the NMJ that ishighlighted in the insets below. At least 100 NMJs was randomly scoredin each muscle per animal. A normal NMJ was defined as having apre-synaptic terminus that did not exhibit the abnormal accumulation ofneurofilament protein shown in FIG. 6A. Values in FIG. 6F represent themean f SEM. Key: WT, untreated wild type; HET, untreated heterozygote;SMA, untreated knockout; AAV, AAVhSMN1-treated SMA mice; *, p<0.05; **,p<0.01; ***, p<0.001. Scale bars: 20 μm.

FIGS. 7A-7F show the results of behavioral tests in treated anduntreated SMA mice. Treated SMA mice showed significant improvements onbehavioral tests. Treated SMA (asterisk) and untreated wild-type (WT)mice were substantially fitter than untreated SMA mice (labeled ‘x’) at16 days (FIG. 7A). Treated SMA mice were also significantly heavier thanuntreated SMA controls from day 11 and onwards (FIG. 7B). Treated SMAmice performed significantly better than untreated SMA mice on therighting reflex (FIG. 7C), negative geotaxis (FIG. 7D), grip strength(FIG. 7E) and hindlimb splay (FIG. 7F) tests. Treated SMA mice werestatistically identical to wild-type and heterozygote mice on therighting reflex and negative geotaxis tests at 12-16 days (FIGS. 7C and7D). Values represent the mean t SEM. Key: untreated WT (open circle),untreated heterozygote (open triangle); untreated SMA (open square);AAVhSMN1-treated SMA mice (closed square); *, p<0.05; **, p<0.01; ***,p<0.001.

FIG. 8 shows survival of scAAVhSMN1-treated and untreated mice.Treatment with scAAVhSMN1 increased survival in SMA mice. SMA micetreated at P0 with scAAVhSMN1 (n=17, closed triangles) had a medianlifespan of 157 days (p<0.0001), compared to 16 days in untreated SMAmice (n=47, open circles).

FIGS. 9A-9B (SEQ ID NOS:1 and 2) show the coding DNA sequence (FIG. 9A)and the corresponding amino acid sequence (FIG. 9B) of a representativehuman survival motor neuron (SMN1) gene.

FIGS. 10A-10F shows that scAAV8-hSMN expression increases motor neuroncounts and improves NMJ in SMA mice. FIG. 10A shows the percentage ofmChAT immunopositive cells that co-localized with hSMN expression in thethoracic-lumbar region at 16 days post-injection. FIGS. 10B-10F show theaverage numbers of mChAT immunopositive cells in the lumbar (FIG. 10B),thoracic (FIG. 10C), and cervical (FIG. 10D) segments, and the averagepercentages of collapsed NMJs in the quadriceps (FIG. 10E) andintercostal (FIG. 10F) muscles at 16, 58-66 and 214-269 days. As areference for FIGS. 10E and 10F, 75-90% of NMJ in the quadriceps andintercostal muscles of untreated SMA mice contained an aberrantcollapsed structure at 16 days (see FIG. 6F). Key and n-values: SMA,untreated knockout (open bars, n=8 at 16 days), AAV, AAV8-hSMN (hatchedbars, n=8 at 16 days, n=5 at 58-66 days); scAAV, scAAV8-hSMN (closedbars, n=5 at each time point); WT, untreated WT (checkered bars, n=8 at16 days, n=5 each at 58-66 and 216-269 days). Values represent the meanf SEM. Statistical comparisons were performed with one-way ANOVA andBonferroni multiple post hoc tests at 16 days (FIGS. 10B-10F). Theunpaired two-tailed student t-test compared 1) the two vectors to eachother at 16 days (FIG. 10A) and 58-66 days (FIGS. 10B-10D); 2) therelative number of ChAT cells in the 58-66d and 214-269d groups withscAAV8-hSMN treatment (FIGS. 10B-10D); 3) the relative number ofabnormal NMJs between the age-matched untreated WT andscAAV8-hSMN-treated SMA mice at 214-269 days (E, F); *p<0.05, **p<0.01,***p<0.001.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, recombinantDNA techniques and immunology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., FundamentalVirology, 2nd Edition, vol. I & II (B. N. Fields and D. M. Knipe, eds.);Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C.Blackwell eds., Blackwell Scientific Publications); T. E. Creighton,Proteins: Structures and Molecular Properties (W.H. Freeman and Company,1993); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., currentaddition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2ndEdition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds.,Academic Press, Inc.).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

1. Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to “an interleukin receptor” includes a mixture of two or moresuch receptors, and the like.

The terms “polypeptide” and “protein,” used interchangeably herein, or anucleotide sequence encoding the same, refer to a protein or nucleotidesequence, respectively, that represents either a native sequence, avariant thereof or a fragment thereof. The full-length proteins, with orwithout the signal sequence, and fragments thereof, as well as proteinswith modifications, such as deletions, additions and substitutions(either conservative or non-conservative in nature), to the nativesequence, are intended for use herein, so long as the protein maintainsthe desired activity. These modifications may be deliberate, as throughsite-directed mutagenesis, or may be accidental, such as throughmutations of hosts which produce the proteins or errors due to PCRamplification. Accordingly, active proteins substantially homologous tothe parent sequence, e.g., proteins with 70 . . . 80 . . . 85 . . . 90 .. . 95 . . . 98 . . . 99% etc. identity that retain the desired activityof the native molecule, are contemplated for use herein.

A “native” polypeptide, such as a survival motor neuron (SMN)polypeptide, refers to a polypeptide having the same amino acid sequenceas the corresponding molecule derived from nature. Such native sequencescan be isolated from nature or can be produced by recombinant orsynthetic means. The term “native” sequence specifically encompassesnaturally-occurring truncated or secreted forms of the specific molecule(e.g., an extracellular domain sequence), naturally-occurring variantforms (e.g., alternatively spliced forms) and naturally-occurringallelic variants of the polypeptide. In various embodiments of theinvention, the native molecules disclosed herein are mature orfull-length native sequences comprising the full-length amino acidssequences shown in the accompanying figures. However, while some of themolecules disclosed in the accompanying figures begin with methionineresidues designated as amino acid position 1 in the figures, othermethionine residues located either upstream or downstream from aminoacid position 1 in the figures may be employed as the starting aminoacid residue for the particular molecule. Alternatively, depending onthe expression system used, the molecules described herein may lack anN-terminal methionine.

By “variant” is meant an active polypeptide as defined herein having atleast about 80% amino acid sequence identity with the correspondingfull-length native sequence, a polypeptide lacking the signal peptide,an extracellular domain of a polypeptide, with or without a signalpeptide, or any other fragment of a full-length polypeptide sequence asdisclosed herein. Such polypeptide variants include, for instance,polypeptides wherein one or more amino acid residues are added, ordeleted, at the N- and/or C-terminus of the full-length native aminoacid sequence. Ordinarily, a variant will have at least about 80% aminoacid sequence identity, alternatively at least about 81% amino acidsequence identity, alternatively at least about 82% amino acid sequenceidentity, alternatively at least about 83% amino acid sequence identity,alternatively at least about 84% amino acid sequence identity,alternatively at least about 85% amino acid sequence identity,alternatively at least about 86% amino acid sequence identity,alternatively at least about 87% amino acid sequence identity,alternatively at least about 88% amino acid sequence identity,alternatively at least about 89% amino acid sequence identity,alternatively at least about 90% amino acid sequence identity,alternatively at least about 91% amino acid sequence identity,alternatively at least about 92% amino acid sequence identity,alternatively at least about 93% amino acid sequence identity,alternatively at least about 94% amino acid sequence identity,alternatively at least about 95% amino acid sequence identity,alternatively at least about 96% amino acid sequence identity,alternatively at least about 97% amino acid sequence identity,alternatively at least about 98% amino acid sequence identity andalternatively at least about 99% amino acid sequence identity to thecorresponding full-length native sequence. Ordinarily, variantpolypeptides are at least about 10 amino acids in length, such as atleast about 20 amino acids in length, e.g., at least about 30 aminoacids in length, alternatively at least about 40 amino acids in length,alternatively at least about 50 amino acids in length, alternatively atleast about 60 amino acids in length, alternatively at least about 70amino acids in length, alternatively at least about 80 amino acids inlength, alternatively at least about 90 amino acids in length,alternatively at least about 100 amino acids in length, alternatively atleast about 150 amino acids in length, alternatively at least about 200amino acids in length, alternatively at least about 300 amino acids inlength, or more.

Particularly preferred variants include substitutions that areconservative in nature, i.e., those substitutions that take place withina family of amino acids that are related in their side chains.Specifically, amino acids are generally divided into four families: (1)acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine;(3) non-polar—alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine,asparagine, glutamine, cysteine, serine threonine, tyrosine.Phenylalanine, tryptophan, and tyrosine are sometimes classified asaromatic amino acids. For example, it is reasonably predictable that anisolated replacement of leucine with isoleucine or valine, an aspartatewith a glutamate, a threonine with a serine, or a similar conservativereplacement of an amino acid with a structurally related amino acid,will not have a major effect on the biological activity. For example,the polypeptide of interest may include up to about 5-10 conservative ornon-conservative amino acid substitutions, or even up to about 15-25 or50 conservative or non-conservative amino acid substitutions, or anynumber between 5-50, so long as the desired function of the moleculeremains intact.

“Homology” refers to the percent identity between two polynucleotide ortwo polypeptide moieties. Two DNA, or two polypeptide sequences are“substantially homologous” to each other when the sequences exhibit atleast about 50%, preferably at least about 75%, more preferably at leastabout 80%-85%, preferably at least about 90%, and most preferably atleast about 95%-98% sequence identity over a defined length of themolecules. As used herein, substantially homologous also refers tosequences showing complete identity to the specified DNA or polypeptidesequence.

In general, “identity” refers to an exact nucleotide-to-nucleotide oramino acid-to-amino acid correspondence of two polynucleotides orpolypeptide sequences, respectively. Percent identity can be determinedby a direct comparison of the sequence information between two moleculesby aligning the sequences, counting the exact number of matches betweenthe two aligned sequences, dividing by the length of the shortersequence, and multiplying the result by 100. Readily available computerprograms can be used to aid in the analysis, such as ALIGN, Dayhoff, M.O. in Atlas of Protein Sequence and Structure M. O. Dayhoffed., 5 Suppl.1:353-358, National Biomedical Research Foundation, Washington, D.C.,which adapts the local homology algorithm of Smith and Waterman Advancesin Appl. Math. 2:482-489, 1981 for peptide analysis. Programs fordetermining nucleotide sequence identity are available in the WisconsinSequence Analysis Package, Version 8 (available from Genetics ComputerGroup, Madison, Wis.) for example, the BESTFIT, FASTA and GAP programs,which also rely on the Smith and Waterman algorithm. These programs arereadily utilized with the default parameters recommended by themanufacturer and described in the Wisconsin Sequence Analysis Packagereferred to above. For example, percent identity of a particularnucleotide sequence to a reference sequence can be determined using thehomology algorithm of Smith and Waterman with a default scoring tableand a gap penalty of six nucleotide positions.

Another method of establishing percent identity in the context of thepresent invention is to use the MPSRCH package of programs copyrightedby the University of Edinburgh, developed by John F. Collins and ShaneS. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View,Calif.). From this suite of packages the Smith-Waterman algorithm can beemployed where default parameters are used for the scoring table (forexample, gap open penalty of 12, gap extension penalty of one, and a gapof six). From the data generated the “Match” value reflects “sequenceidentity.” Other suitable programs for calculating the percent identityor similarity between sequences are generally known in the art, forexample, another alignment program is BLAST, used with defaultparameters. For example, BLASTN and BLASTP can be used using thefollowing default parameters: genetic code=standard; filter=none;strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swissprotein+Spupdate+PIR. Details of these programs are well known in theart.

Alternatively, homology can be determined by hybridization ofpolynucleotides under conditions which form stable duplexes betweenhomologous regions, followed by digestion with single-stranded-specificnuclease(s), and size determination of the digested fragments. DNAsequences that are substantially homologous can be identified in aSouthern hybridization experiment under, for example, stringentconditions, as defined for that particular system. Defining appropriatehybridization conditions is within the skill of the art. See, e.g.,Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization,supra.

By the term “degenerate variant” is intended a polynucleotide containingchanges in the nucleic acid sequence thereof, that encodes a polypeptidehaving the same amino acid sequence as the polypeptide encoded by thepolynucleotide from which the degenerate variant is derived.

A “coding sequence” or a sequence which “encodes” a selectedpolypeptide, is a nucleic acid molecule which is transcribed (in thecase of DNA) and translated (in the case of mRNA) into a polypeptide invivo when placed under the control of appropriate regulatory sequences.The boundaries of the coding sequence are determined by a start codon atthe 5′(amino) terminus and a translation stop codon at the 3′ (carboxy)terminus. A transcription termination sequence may be located 3′ to thecoding sequence.

By “vector” is meant any genetic element, such as a plasmid, phage,transposon, cosmid, chromosome, virus, virion, etc., which is capable ofreplication when associated with the proper control elements and whichcan transfer gene sequences to cells. Thus, the term includes cloningand expression vehicles, as well as viral vectors.

By “recombinant vector” is meant a vector that includes a heterologousnucleic acid sequence which is capable of expression in vivo.

By “recombinant virus” is meant a virus that has been geneticallyaltered, e.g., by the addition or insertion of a heterologous nucleicacid construct into the particle.

The term “transgene” refers to a polynucleotide that is introduced intoa cell and is capable of being transcribed into RNA and optionally,translated and/or expressed under appropriate conditions. In one aspect,it confers a desired property to a cell into which it was introduced, orotherwise leads to a desired therapeutic or diagnostic outcome.

The terms “genome particles (gp),” or “genome equivalents,” as used inreference to a viral titer, refer to the number of virions containingthe recombinant AAV DNA genome, regardless of infectivity orfunctionality. The number of genome particles in a particular vectorpreparation can be measured by procedures such as described in theExamples herein, or for example, in Clark et al., Hum. Gene Ther. (1999)10:1031-1039; and Veldwijk et al., Mol. Ther. (2002) 6:272-278.

The terms “infection unit (iu),” “infectious particle,” or “replicationunit,” as used in reference to a viral titer, refer to the number ofinfectious recombinant AAV vector particles as measured by theinfectious center assay, also known as replication center assay, asdescribed, for example, in McLaughlin et al., J. Virol. (1988)62:1963-1973.

The term “transducing unit (tu)” as used in reference to a viral titer,refers to the number of infectious recombinant AAV vector particles thatresult in the production of a functional transgene product as measuredin functional assays such as described in Examples herein, or forexample, in Xiao et al., Exp. Neurobiol. (1997) 144:1 13-124; or inFisher et al., J. Virol. (1996) 70:520-532 (LFU assay).

The term “transfection” is used to refer to the uptake of foreign DNA bya cell, and a cell has been “transfected” when exogenous DNA has beenintroduced inside the cell membrane. A number of transfection techniquesare generally known in the art. See, e.g., Graham et al. (1973)Virology, 1:456, Sambrook et al. (1989) Molecular Cloning, a laboratorymanual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986)Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene1:197. Such techniques can be used to introduce one or more exogenousDNA moieties into suitable host cells.

The term “heterologous” as it relates to nucleic acid sequences such ascoding sequences and control sequences, denotes sequences that are notnormally joined together, and/or are not normally associated with aparticular cell. Thus, a “heterologous” region of a nucleic acidconstruct or a vector is a segment of nucleic acid within or attached toanother nucleic acid molecule that is not found in association with theother molecule in nature. For example, a heterologous region of anucleic acid construct could include a coding sequence flanked bysequences not found in association with the coding sequence in nature.Another example of a heterologous coding sequence is a construct wherethe coding sequence itself is not found in nature (e.g., syntheticsequences having codons different from the native gene). Similarly, acell transformed with a construct which is not normally present in thecell would be considered heterologous for purposes of this invention.Allelic variation or naturally occurring mutational events do not giverise to heterologous DNA, as used herein.

A “nucleic acid” sequence refers to a DNA or RNA sequence. The termcaptures sequences that include any of the known base analogues of DNAand RNA such as, but not limited to 4-acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxyl-methyl) uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudo-uracil,1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine,2-methylguanine, 3-methyl-cytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxy-amino-methyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, -uracil-5-oxyacetic acid methylester, uracil-5-oxyaceticacid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.

The term DNA “control sequences” refers collectively to promotersequences, polyadenylation signals, transcription termination sequences,upstream regulatory domains, origins of replication, internal ribosomeentry sites (“IRES”), enhancers, and the like, which collectivelyprovide for the replication, transcription and translation of a codingsequence in a recipient cell. Not all of these control sequences needalways be present so long as the selected coding sequence is capable ofbeing replicated, transcribed and translated in an appropriate hostcell.

The term “promoter” is used herein in its ordinary sense to refer to anucleotide region comprising a DNA regulatory sequence, wherein theregulatory sequence is derived from a gene which is capable of bindingRNA polymerase and initiating transcription of a downstream(3′-direction) coding sequence. Transcription promoters can include“inducible promoters” (where expression of a polynucleotide sequenceoperably linked to the promoter is induced by an analyte, cofactor,regulatory protein, etc.), “repressible promoters” (where expression ofa polynucleotide sequence operably linked to the promoter is induced byan analyte, cofactor, regulatory protein, etc.), and “constitutivepromoters”.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, control sequences operably linked to a coding sequenceare capable of effecting the expression of the coding sequence. Thecontrol sequences need not be contiguous with the coding sequence, solong as they function to direct the expression thereof. Thus, forexample, intervening untranslated yet transcribed sequences can bepresent between a promoter sequence and the coding sequence and thepromoter sequence can still be considered “operably linked” to thecoding sequence.

The term “nervous system” includes both the central nervous system andthe peripheral nervous system. The term “central nervous system” or“CNS” includes all cells and tissue of the brain and spinal cord of avertebrate. The term “peripheral nervous system” refers to all cells andtissue of the portion of the nervous system outside the brain and spinalcord. Thus, the term “nervous system” includes, but is not limited to,neuronal cells, glial cells, astrocytes, cells in the cerebrospinalfluid (CSF), cells in the interstitial spaces, cells in the protectivecoverings of the spinal cord, epidural cells (i.e., cells outside of thedura mater), cells in non-neural tissues adjacent to or in contact withor innervated by neural tissue, cells in the epineurium, perineurium,endoneurium, funiculi, fasciculi, and the like.

“Active” or “activity” for purposes of the present invention refers toforms of a therapeutic protein which retain a biological activity of thecorresponding native or naturally occurring polypeptide. The activitymay be greater than, equal to, or less than that observed with thecorresponding native or naturally occurring polypeptide.

By “isolated” when referring to a nucleotide sequence, is meant that theindicated molecule is present in the substantial absence of otherbiological macromolecules of the same type. Thus, an “isolated nucleicacid molecule which encodes a particular polypeptide” refers to anucleic acid molecule which is substantially free of other nucleic acidmolecules that do not encode the subject polypeptide; however, themolecule may include some additional bases or moieties which do notdeleteriously affect the basic characteristics of the composition.

For the purpose of describing the relative position of nucleotidesequences in a particular nucleic acid molecule throughout the instantapplication, such as when a particular nucleotide sequence is describedas being situated “upstream,” “downstream,” “3-prime (3′)” or “5-prime(5′)” relative to another sequence, it is to be understood that it isthe position of the sequences in the “sense” or “coding” strand of a DNAmolecule that is being referred to as is conventional in the art.

The term “about”, particularly in reference to a given quantity, ismeant to encompass deviations of plus or minus five percent.

The terms “subject”, “individual” or “patient” are used interchangeablyherein and refer to a vertebrate, preferably a mammal. Mammals include,but are not limited to, murines, rodents, simians, humans, farm animals,sport animals and pets.

The term “modulate” as used herein means to vary the amount or intensityof an effect or outcome, e.g., to enhance, augment, diminish, reduce oreliminate.

As used herein, the term “ameliorate” is synonymous with “alleviate” andmeans to reduce or lighten. For example, one may ameliorate the symptomsof a disease or disorder by making the disease or symptoms of thedisease less severe.

The terms “therapeutic,” “effective amount” or “therapeuticallyeffective amount” of a composition or agent, as provided herein, referto a sufficient amount of the composition or agent to provide thedesired response, such as the prevention, delay of onset or ameliorationof symptoms in a subject or an attainment of a desired biologicaloutcome, such as correction of neuropathology, e.g., cellular pathologyassociated with a motor neuronal disease such as spinal muscular atrophy(SMA). The term “therapeutic correction” refers to that degree ofcorrection that results in prevention or delay of onset or ameliorationof symptoms in a subject. The exact amount required will vary fromsubject to subject, depending on the species, age, and general conditionof the subject, the severity of the condition being treated, and theparticular macromolecule of interest, mode of administration, and thelike. An appropriate “effective” amount in any individual case may bedetermined by one of ordinary skill in the art using routineexperimentation.

“Treatment” or “treating” a particular disease includes: (1) preventingthe disease, i.e. preventing the development of the disease or causingthe disease to occur with less intensity in a subject that may beexposed to or predisposed to the disease but does not yet experience ordisplay symptoms of the disease, (2) inhibiting the disease, i.e.,arresting the development or reversing the disease state, or (3)relieving symptoms of the disease i.e., decreasing the number ofsymptoms experienced by the subject, as well as changing the cellularpathology associated with the disease.

2. Modes of Carrying Out the Invention

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular formulationsor process parameters as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments of the invention only, and is notintended to be limiting.

Although a number of methods and materials similar or equivalent tothose described herein can be used in the practice of the presentinvention, the preferred materials and methods are described herein.

Central to the present invention is the discovery that delivery of rAAVvirions containing the human survival motor neuron 1 (hSMN1) cDNA to theCNS of an aggressive mouse model of spinal muscular atrophy (SMA),produced expression of SMN1 throughout the spinal cord. Treated SMA micecontained a higher number of motor neurons compared to untreated,age-matched mutants. In addition, the evaluation of myofiber sizedemonstrated that the size of individual myofibers from a variety ofmuscle groups in treated SMA mice approximated those observed inwild-type mice. Furthermore, the structure of the neuromuscular junction(NMJ) in treated SMA mice was similar to wild-type mice, which was incontrast to untreated SMA that showed abnormal accumulation ofneurofilament protein at the pre-synaptic termini. Treated SMA mice alsodisplayed significant improvements on a battery of behavioral testssuggesting that the NMJ was functional. Importantly, recombinant AAVtreated mice had a significantly increased lifespan as compared to theiruntreated counterparts. SMA mice treated with a self-complementary rAAVvector also displayed a remarkable improvement in median survival, evenas compared to treatment with conventional, non-self-complementary rAAVvectors.

These results demonstrate that CNS-directed, AAV-mediated SMN1 geneaugmentation is highly efficacious in addressing both the neuronal andmuscular pathologies of SMA and evidence the utility of viral genetherapy as a therapeutic strategy for treating and preventing neuronaland muscular pathologies, such as SMA, as well as other diseases thataffect motor function. The gene therapy techniques described herein canbe used alone, or in conjunction with traditional drugs.

In order to further an understanding of the invention, a more detaileddiscussion is provided below regarding motor neuron pathologies andtherapeutic molecules, as well as various gene delivery methods for usewith the present invention.

Motor Neuron Pathologies and Therapeutic Molecules

The subject invention provides compositions and methods to modulate,correct or augment motor function in a subject afflicted with a motorneuron disorder or with motor neuronal damage. For the purpose ofillustration only, the subject may suffer from one or more of spinalmuscular atrophy (SMA), amytrophic lateral sclerosis (ALS), spinalbulbar muscular atrophy, spinal cerebellar ataxia, primary lateralsclerosis (PLS), or traumatic spinal cord injury. Without being bound bya particular theory, the pathology associated with motor neuron damagemay include motor neuron degeneration, gliosis, neurofilamentabnormalities, loss of myelinated fibers in corticospinal tracts andventral roots. For example, two types of onset have beenrecognized—bulbar onset, which affects the upper motor neurons (cortexand brainstem motor neurons), affects the facial muscles, speech, andswallowing; and limb onset, which affects the lower motor neurons(spinal cord motor neurons), is reflected by spasticity, generalizedweakness, muscular atrophy, paralysis, and respiratory failure. In ALS,subjects have both bulbar and limb onset. In PLS, subjects just havebulbar onset.

Thus, in certain embodiments, the subject is provided with rAAVconstructs that encode a biologically active molecule, the expression ofwhich in the CNS results in at least partial correction ofneuropathology and/or stabilization of disease progression, such as theprevention, delay of onset or amelioration of symptoms in a subject oran attainment of a desired biological outcome, including for example, achange in the cellular pathology associated with a motor neuronaldisease described above.

By way of example, the transgene present in the rAAV construct may be,but is not limited to, survival motor neuron protein (via the SMN1 geneor SMN2 gene), insulin growth factor-1 (IGF-1), calbindin D28,parvalbumin, HIFI-alpha, SIRT-2, VEGF such as VEGF165, CNTF (Ciliaryneurotrophic factor), sonic hedgehog (shh), erythropoietin (EPO), lysyloxidase (LOX), progranulin, prolactin, ghrelin, neuroserpin, angiogenin,and placenta lactogen.

The molecular basis of SMA, an autosomal recessive neuromusculardisorder, is the homozygous loss of the survival motor neuron gene 1(SMN1), which may also be known as SMN Telomeric. A nearly identicalcopy of the SMN1 gene, called SMN2, which may also be known as SMNCentromeric, is found in humans and modulates the disease severity.Expression of the normal SMN1 gene results solely in expression ofsurvival motor neuron (SMN) protein. Expression of the SMN2 gene resultsin approximately 10-20% of the SMN protein and 80-90% of anunstable/non-functional SMNdelta7 protein. Only 10% of SMN2 transcriptsencode a functional full-length protein identical to SMN1. Thisfunctional difference between both genes results from a translationallysilent mutation that, however, disrupts an exonic splicing enhancercausing exon 7 skipping in most SMN2 transcripts. SMN protein plays awell-established role in assembly of the spliceosome and may alsomediate mRNA trafficking in the axon and nerve terminus of neurons.

The nucleotide and amino acid sequences of various SMN1 molecules andSMN proteins are known. See, for example, FIGS. 9A-9B; NCBI accessionnumbers NM_000344 (human), NP_000335 (human), NM_011420 (mouse), EU791616 (porcine), NM_001131470 (orangutan), NM_131191 (zebrafish),BC062404 (rat), NM_001009328 (cat), NM_001003226 (dog), NM_175701 (cow).Similarly, various SMN2 sequences are known. See, e.g., NCBI accessionnumbers NM_022876, NM_022877, NM_017411, NG_008728, BC_000908, BC070242,DQ185039 (all human).

Insulin-like growth factor 1 (IGF-I) is a therapeutic protein for thetreatment of neurodegenerative disorders, including motor neurondisorders, due to its many actions at different levels of neuraxis (seeDore et al., Trends Neurosci (1997)₂₀:326-331). For example, in thebrain it is thought to reduce both neuronal and glial apoptosis, protectneurons against toxicity induced by iron, colchicine, calciumdestabilizers, peroxides, and cytokines. It also appears to modulate therelease of neurotransmitters acetylcholine and glutamate and induce theexpression of neurofilament, tublin, and myelin basic protein. In thespinal cord, IGF-I is believed to modulate ChAT activity and attenuateloss of cholinergic phenotype, enhance motor neuron sprouting, increasemyelination, inhibit demyelination, stimulate motor neuron proliferationand differentiation from precursor cells, and promote Schwann celldivision, maturation, and growth. In the muscle, IGF-I appears to induceacetylcholine receptor cluster formation at the neuromuscular junctionand increase neuromuscular function and muscle strength.

The IGF-1 gene has a complex structure, which is well-known in the art.It has at least two alternatively spliced mRNA products arising from thegene transcript. There is a 153 amino acid peptide, known by severalnames including IGF-IA or IGF-IEa, and a 195 amino acid peptide, knownby several names including IGF-IB or IGF-IEb. The Eb form is also beknown as Ec in humans. The mature form of IGF-I is a 70 amino acidpolypeptide. Both IGF-IEa and IGF-IEb contain the 70 amino acid maturepeptide, but differ in the sequence and length of theircarboxyl-terminal extensions. The IGF-1 proteins, as well as the peptidesequences of IGF-IEa and IGF-IEb are known and described in, e.g.,International Publication No. WO 2007/146046, incorporated herein byreference in its entirety. The genomic and functional cDNAs of humanIGF-I, as well as additional information regarding the IGF-I gene andits products, are available at Unigene Accession No. NM_000618.

Calbindin D28K (also referred to as calbindin D28) and parvalbumin arecalcium-binding proteins believed to be involved in calcium buffering.Without being bound by a particular theory, calcium homeostasis appearsto be altered in subjects with motor neuron disorders (e.g., ALS) andlow levels of calbindin-D28K and/or parvalbumin may increase thevulnerability of motor neurons by reducing their ability to handle anincreased calcium load. This reduction may lead to cell injury andeventual motor neuron death. Further evidence suggests that neurons richin calcium-binding proteins, such as calbindin D28K and parvalbumin, areresistant to degeneration.

HIF-I is a heterodimeric protein composed of two subunits: (i) aconstitutively expressed β subunit also known as aryl hydrocarbonnuclear translocator (ARNT) (which is shared by other relatedtranscription factors (e.g., the dioxin/aryl hydrocarbon receptor(DR/AhR)); and (ii) an a subunit (see, e.g., International publicationNo. WO 96/39426, describing the recent affinity purification andmolecular cloning of HIF-Iα. Both subunits are members of the basichelix-loop-helix (bHLH)-PAS family of transcription factors. Thesedomains regulate DNA binding and dimerization. The transactivationdomain resides in the C-terminus of the protein. The basic regionconsists of approximately 15 predominantly basic amino acids responsiblefor direct DNA binding. This region is adjacent to two amphipathic ahelices, separated by a loop of variable length, which forms the primarydimerization interface between family members (Moore, et al., Proc.Natl. Acad Sci. USA (2000) 97:10436-10441). The PAS domain encompasses200-300 amino acids containing two loosely conserved, largelyhydrophobic regions approximately 50 amino acids, designated PAS A andPAS B. The HIF-Iα subunit is unstable during normoxic conditions,overexpression of this subunit in cultured cells under normal oxygenlevels is capable of inducing expression of genes normally induced byhypoxia. Replacement of the C terminal (or transactivation) region ofthe hypoxia-inducible factor protein with a strong transactivationdomain from a transcriptional activator protein such as, for example,Herpes Simplex Virus (HSV) VP16, NFκB or yeast transcription factorsGAL4 and GCN4, is designed to stabilize the protein under normoxicconditions and provide strong, constitutive, transcriptional activation.See, e.g., International Publication No. WO 2008/042420 for adescription and sequence of a representative stabilizedhypoxia-inducible factor protein that is a hybrid/chimeric fusionprotein consisting of the DNA-binding and dimerization domains fromHIF-la and the transactivation domain from the HSV VP16 protein,incorporated herein by reference in its entirety. See, also, U.S. Pat.Nos. 6,432,927 and 7,053,062 for a description of a constitutivelystable hybrid HIF-Iα, both of which are incorporated by reference hereinin their entirety,

Members of the vascular endothelial growth factor (VEGF) family areamong the most powerful modulators of vascular biology. They regulatevasculogenesis, angiogenesis, and vascular maintenance. Four differentmolecular variants of VEGF have been described. The 165 amino acidvariant is the predominant molecular form found in normal cells andtissues. A less abundant, shorter form with a deletion of 44 amino acidsbetween positions 116 and 159 (VEGF₁₂₁), a longer form with an insertionof 24 basic residues in position 116 (VEGF₁₈₉), and another longer formwith an insertion of 41 amino acids (VEGF₂₀₆), which includes the 24amino acid insertion found in VEGF₁₈₉, are also known. VEGF₁₂₁ andVEGF₁₆₅ are soluble proteins. VEGF₁₈₉ and VEGF₂₀₆ appear to be mostlycell-associated. All of the versions of VEGF are biologically active.See, e.g., Tischer et al., J. Biol. Chem. (1991) 266:11947-11954,describing the sequence of VEGF₁₆₅ (see, also, GenBank Accession no.AB021221), VEGF₁₂₁ (see, also, GenBank Accession no. AF214570) andVEGF₁₈₉; and Houck et al., Mol. Endocrinol. (1991) 5:1806-1814,describing the sequence of VEGF₂₀₆.

CNTF (Ciliary neurotrophic factor) is a neurocytokine expressed by glialcells in peripheral nerves and the central nervous system. CNTF isgenerally recognized for its function in support and survival ofnon-neuronal and neuronal cell types. See e.g., Vergara, C and Ramirez,B; Brain Res, Brain Res. Rev. (2004) 47; 161-73.

Sonic hedgehog (Shh) controls important developmental processes,including neuronal and glial cell survival.

Erythropoietin (EPO) is a principal regulator of erythroid progenitorcells. However, it is functionally expressed in the nervous system andhas been reported to have a neuroprotective effects. See e.g.,Bartesaghi, S., 2005. Neurotoxicology, 26:923-8. Genes encoding humanand other mammalian EPO have been cloned, sequenced and expressed, andshow a high degree of sequence homology in the coding region acrossspecies. Wen et al., Blood (1993) 82:1507-1516. The sequence of the geneencoding native human EPO, as well as methods of obtaining the same, aredescribed in, e.g., U.S. Pat. Nos. 4,954,437 and 4,703,008, incorporatedherein by reference in their entirety, as well as in Jacobs et al.(1985) Nature 313:806-810; Lin et al. (1985) Proc. Natl. Acad. Sci. USA0.2:7580; International Publication Number WO 85/02610; and EuropeanPatent Publication Number 232,034 B1. In addition, the sequences of thegenes encoding native feline, canine and porcine EPO are known andreadily available (GenBank Accession Nos.: L10606; L13027; and L10607,respectively), and the sequence of the gene encoding monkey (Macacamulatta) is also known and available (GenBank Accession No.: L10609).

Lysyl oxidase (LOX) oxidizes the side chain of peptidyl lysine therebyconverting certain lysine residues toalpha-aminoadipic-delta-semialdehyde. This is a post-translationalchange that, for example, enables the covalent cross-linking of thecomponent chains of collagen and elastin. It stabilizes the fibrousdeposits of these proteins in the extracellular matrix. LOX can alsooxidize lysine within a variety of cationic proteins, which suggeststhat its functions are broader than stabilization or the extracellularmatrix. LOX is synthesized as a preprotein; it emerges from the cell asproLOX and is processed proteolytically to the active enzyme. See e.g.,Lucero, H A and Kagan, H M, Cell MoI. Life Sci. (2006) 3:2304-2316.

Progranulin (PGRN) is a pleitropic protein. Mutations in the gene causefrontotemporal lobar degeneration. PGRN in the CNS is expressed bymicroglia and neurons and plays a role in brain development. PGRN isalso involved in multiple “tissue modeling” processes includingdevelopment, wound repair and tumorogenesis. PGRN is converted toGranulin (GRN) by elastase enzymes. While progranulin has trophicproperties, GRNs are more akin to inflammatory mediators. Geneexpression studies from animal models of CNS disease show a differentialincrease in PRGN combined with microglial activation and inflammation.Increase in PGRN expression may be closely related to microglialactivation and neuroinflammation. Moreover, PGRN expression is increasedin activated microglia in many neurodegenerative diseases includingmotor neuron disease and Alzheimer's disease. Studies have identifiedmutations in PGRN as a cause of neurodegenerative disease and indicatethe importance of PGRN function for neuronal survival.

Oligodendrocytes, the myelinating cells of the CNS, continue to begenerated by oligodendrocyte precursor cells (OPCs) throughout adulthoodand are required for the intrinsic repair of myelin damage in the adultCNS. The physiological events that modulate OPC proliferation and thegeneration of new myelinating oligodendrocytes in the adult CNS arelargely known. Recently it has been reported that patients with MultipleSclerosis (MS), a demyleinating disease, have a reduced relapse rateduring the third trimester of pregnancy suggesting that hormonesinfluence oligodendrocyte generation. Remission in MS patients iscorrelated with a decrease in the number and size of active white matterlesions. Pregnancy in mice results in an increase in the generation ofnew oligodendrocytes and the number of myelinated axons within thematernal CNS (Gregg et al., J. Neurosci. (2007) 22:1812-1823).Prolactin, a hormone that plateaus during the final stage of pregnancy,has been shown to regulate OPC proliferation during pregnancy andpromote white matter repair in virgin female mice (Gregg et al., J.Neurosci. (2007) 27:1812-1823).

Human placenta lactogen (hPL), a hormone that also peaks during thethird trimester of pregnancy may have a similar influence onoligodendrocyte generation. hPL has a number of biological activitiesthat are qualitatively similar to human growth hormone (hGH) andprolactin and appears to be a major regulator of IGF-I production. BothhGH and IGF-I have been shown to be stimulators of myelination in theadult CNS (Carson et al., Neuron (1993) 10:729-740; Peltwon et al.,Neurology (1977) 22:282-288). Therefore, the treatment of CNS diseasesinvolving demyelination such as MS, ALS, stroke and spinal cord injurymay benefit from PRL- or hPL-based therapies, such as by theintraventricular injection of an rhPRL or hPL expressing viral vector.

Ghrelin is a gastric hormone that is a mediator of growth hormonerelease. See e.g. Wu, et al., Ann. Surg. (2004) 239:464.

Neuroserpin is a serpin protease inhibitor family member. In certain CNSconditions, neuroserpin can play a neuroprotective role potentiallythrough the blockage of the effects of tPA. See, e.g., Galliciotti, Gand Sonderegger, P, Front Biosci (2006) 11:33; Simonin, et al., (2006)26:10614; Miranda, E and Lomas, D A, Cell Mol Life Sci (2006) 63:709.

Angiogenin is a member of the RNAse superfamily. It is a normalconstituent of circulation but has also been implicated as a risk factorin motor neuron disorders.

In certain compositions and methods of the invention, more than onetransgene encoding more than one of the therapeutic molecules describedabove can be delivered, wherein each transgene is operably linked to apromoter to enable the expression of the trangenes from a single AAVvector. In additional methods, the transgenes may be operably linked tothe same promoter. Each transgene encodes a biologically activemolecule, expression of which in the CNS results in at least partialcorrection of neuropathology. Additionally, in cases where more than onetransgene is delivered, the transgenes may be delivered via more thanone AAV vector, wherein each AAV vector comprises a transgene operablylinked to a promoter.

The native molecules, as well as active fragments and analogs thereof,which retain the desired biological activity, as measured in any of thevarious assays and animal models including those described furtherherein, are intended for use with the present invention.

Polynucleotides encoding the desired protein for use with the presentinvention can be made using standard techniques of molecular biology.For example, polynucleotide sequences coding for the above-describedmolecules can be obtained using recombinant methods, such as byscreening cDNA and genomic libraries from cells expressing the gene, orby deriving the gene from a vector known to include the same. The geneof interest can also be produced synthetically, rather than cloned,based on the known sequences. The molecules can be designed withappropriate codons for the particular sequence. The complete sequence isthen assembled from overlapping oligonucleotides prepared by standardmethods and assembled into a complete coding sequence. See, e.g., Edge,Nature (1981) 292:756; Nambair et al., Science (1984) 22:1299; and Jayet al., J. Biol. Chem. (1984) 22:6311.

Thus, particular nucleotide sequences can be obtained from vectorsharboring the desired sequences or synthesized completely or in partusing various oligonucleotide synthesis techniques known in the art,such as site-directed mutagenesis and polymerase chain reaction (PCR)techniques where appropriate. See, e.g., Sambrook, supra. One method ofobtaining nucleotide sequences encoding the desired sequences is byannealing complementary sets of overlapping synthetic oligonucleotidesproduced in a conventional, automated polynucleotide synthesizer,followed by ligation with an appropriate DNA ligase and amplification ofthe ligated nucleotide sequence via PCR. See, e.g., Jayaraman et al.,Proc. Natl. Acad. Sci. USA (1991) 88:4084-4088. Additionally,oligonucleotide-directed synthesis (Jones et al., Nature (1986)54:75-82), oligonucleotide directed mutagenesis of preexistingnucleotide regions (Riechmann et al., Nature (1988) 332:323-327 andVerhoeyen et al., Science (1988) 2& 1534-1536), and enzymatic filling-inof gapped oligonucleotides using T₄ DNA polymerase (Queen et al., Proc.Natl. Acad. Sci. USA (1989) 6:10029-10033) can be used to providemolecules for use in the subject methods.

Once produced, the constructs are delivered using recombinant viralvectors as described further below.

AAV Gene Delivery Techniques

The constructs described above, are delivered to the subject in questionusing any of several rAAV gene delivery techniques. Several AAV-mediatedmethods for gene delivery are known in the art. As described furtherbelow, genes can be delivered either directly to the subject or,alternatively, delivered ex vivo, to appropriate cells, such as cellsderived from the subject, and the cells reimplanted in the subject.

Various AAV vector systems have been developed for gene delivery. AAVvectors can be readily constructed using techniques well known in theart. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; InternationalPublication Nos. WO 92/01070 (published 23 Jan. 1992) and WO 93/03769(published 4 Mar. 1993); Lebkowski et al., Molec. Cell. Biol. (1988)1:3988-3996; Vincent et al., Vaccines 90 (1990) (Cold Spring HarborLaboratory Press); Carter, B. J. Current Opinion in Biotechnology (1992)3:533-539; Muzyczka, N. Current Topics in Microbiol. and Immunol. (1992)158:97-129; Kotin, R. M. Human Gene Therapy (1994) 5:793-801; Shellingand Smith, Gene Therapy (1994) 1:165-169; and Zhou et al., J. Exp. Med.(1994) 179:1867-1875. AAV vector systems are also described in furtherdetail below.

The AAV genome is a linear, single-stranded DNA molecule containingabout 4681 nucleotides. The AAV genome generally comprises an internal,nonrepeating genome flanked on each end by inverted terminal repeats(ITRs). The ITRs are approximately 145 base pairs (bp) in length. TheITRs have multiple functions, including providing origins of DNAreplication, and packaging signals for the viral genome. The internalnonrepeated portion of the genome includes two large open readingframes, known as the AAV replication (rep) and capsid (cap) genes. Therep and cap genes code for viral proteins that allow the virus toreplicate and package into a virion. In particular, a family of at leastfour viral proteins are expressed from the AAV rep region, Rep 78, Rep68, Rep 52, and Rep 40, named according to their apparent molecularweight. The AAV cap region encodes at least three proteins, VPI, VP2,and VP3.

AAV has been engineered to deliver genes of interest by deleting theinternal nonrepeating portion of the AAV genome (i.e., the rep and capgenes) and inserting a heterologous gene between the ITRs. Theheterologous gene is typically functionally linked to a heterologouspromoter (constitutive, cell-specific, or inducible) capable of drivinggene expression in the patient's target cells under appropriateconditions. Examples of each type of promoter are well-known in the art.Termination signals, such as polyadenylation sites, can also beincluded.

AAV is a helper-dependent virus; that is, it requires coinfection with ahelper virus (e.g., adenovirus, herpesvirus or vaccinia), in order toform AAV virions. In the absence of coinfection with a helper virus, AAVestablishes a latent state in which the viral genome inserts into a hostcell chromosome, but infectious virions are not produced. Subsequentinfection by a helper virus “rescues” the integrated genome, allowing itto replicate and package its genome into an infectious AAV virion. WhileAAV can infect cells from different species, the helper virus must be ofthe same species as the host cell. Thus, for example, human AAV willreplicate in canine cells coinfected with a canine adenovirus.

Recombinant AAV virions comprising the gene of interest may be producedusing a variety of art-recognized techniques described more fully below.Wild-type AAV and helper viruses may be used to provide the necessaryreplicative functions for producing rAAV virions (see, e.g., U.S. Pat.No. 5,139,941, incorporated herein by reference in its entirety).Alternatively, a plasmid, containing helper function genes, incombination with infection by one of the well-known helper viruses canbe used as the source of replicative functions (see e.g., U.S. Pat. Nos.5,622,856 and 5,139,941, both incorporated herein by reference in theirentireties). Similarly, a plasmid, containing accessory function genescan be used in combination with infection by wild-type AAV, to providethe necessary replicative functions. These three approaches, when usedin combination with a rAAV vector, are each sufficient to produce rAAVvirions. Other approaches, well known in the art, can also be employedby the skilled artisan to produce rAAV virions.

In one embodiment of the present invention, a triple transfection method(described in detail in U.S. Pat. No. 6,001,650, incorporated byreference herein in its entirety) is used to produce rAAV virionsbecause this method does not require the use of an infectious helpervirus, enabling rAAV virions to be produced without any detectablehelper virus present. This is accomplished by use of three vectors forrAAV virion production: an AAV helper function vector, an accessoryfunction vector, and a rAAV expression vector. One of skill in the artwill appreciate, however, that the nucleic acid sequences encoded bythese vectors can be provided on two or more vectors in variouscombinations.

As explained herein, the AAV helper function vector encodes the “AAVhelper function” sequences (i.e., rep and cap), which function in transfor productive AAV replication and encapsidation. Preferably, the AAVhelper function vector supports efficient AAV vector production withoutgenerating any detectable wt AAV virions (i.e., AAV virions containingfunctional rep and cap genes). An example of such a vector, pHLP19, isdescribed in U.S. Pat. No. 6,001,650, incorporated herein by referencein its entirety. The rep and cap genes of the AAV helper function vectorcan be derived from any of the known AAV serotypes, as explained above.For example, the AAV helper function vector may have a rep gene derivedfrom AAV-2 and a cap gene derived from AAV-6; one of skill in the artwill recognize that other rep and cap gene combinations are possible,the defining feature being the ability to support rAAV virionproduction.

The accessory function vector encodes nucleotide sequences fornon-AAV-derived viral and/or cellular functions upon which AAV isdependent for replication (i.e., “accessory functions”). The accessoryfunctions include those functions required for AAV replication,including, without limitation, those moieties involved in activation ofAAV gene transcription, stage specific AAV mRNA splicing, AAV DNAreplication, synthesis of cap expression products, and AAV capsidassembly. Viral-based accessory functions can be derived from any of thewell-known helper viruses such as adenovirus, herpesvirus, and vacciniavirus. In one embodiment, the accessory function plasmid pLadeno5 isused (details regarding pLadeno5 are described in U.S. Pat. No.6,004,797, incorporated herein by reference in its entirety). Thisplasmid provides a complete set of adenovirus accessory functions forAAV vector production, but lacks the components necessary to formreplication-competent adenovirus.

In order to further an understanding of AAV, a more detailed discussionis provided below regarding recombinant AAV expression vectors and AAVhelper and accessory functions

Recombinant AAV Expression Vectors

Recombinant AAV (rAAV) expression vectors are constructed using knowntechniques to at least provide as operatively linked components in thedirection of transcription, control elements including a transcriptionalinitiation region, the polynucleotide of interest and a transcriptionaltermination region. The control elements are selected to be functionalin the cell of interest, such as in a mammalian cell. The resultingconstruct which contains the operatively linked components is bounded(5′ and 3′) with functional AAV ITR sequences.

The nucleotide sequences of AAV ITR regions are known. See, e.g., Kotin,R. M. (1994) Human Gene Therapy 5:793-801; Berns, K. I. “Parvoviridaeand their Replication” in Fundamental Virology, 2nd Edition, (B. N.Fields and D. M. Knipe, eds.) for the AAV-2 sequence. AAV ITRs used inthe vectors of the invention need not have a wild-type nucleotidesequence, and may be altered, e.g., by the insertion, deletion orsubstitution of nucleotides. Additionally, AAV ITRs may be derived fromany of several AAV serotypes, including without limitation, AAV-1,AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, etc.Furthermore, 5′ and 3′ ITRs which flank a selected nucleotide sequencein an AAV expression vector need not necessarily be identical or derivedfrom the same AAV serotype or isolate, so long as they function asintended, i.e., to allow for excision and rescue of the sequence ofinterest from a host cell genome or vector, and to allow integration ofthe DNA molecule into the recipient cell genome when AAV Rep geneproducts are present in the cell.

Suitable polynucleotide molecules for use in traditional AAV vectorswill be less than or about 5 kilobases (kb) in size. The selectedpolynucleotide sequence is operably linked to control elements thatdirect the transcription or expression thereof in the subject in vivo.Such control elements can comprise control sequences normally associatedwith the selected gene. Alternatively, heterologous control sequencescan be employed. Useful heterologous control sequences generally includethose derived from sequences encoding mammalian or viral genes. Nonlimiting examples of promoters include, but are not limited to, thecytomegalovirus (CMV) promoter (Kaplitt et al., Nat. Genet. (1994)8:148-154), CMV/human β3-globin promoter (Mandel et al., J. Neurosci.(1998) 18:4271-4284), GFAP promoter (Xu et al., Gene Ther. (2001)8:1323-1332), the 1.8-kb neuron-specific enolase (NSE) promoter (Kleinet al., Exp. Neurol. (1998) 150:183-194), chicken beta actin (CBA)promoter (Miyazaki, Gene (1989) 79; 269-277), the β-glucuronidase (GUSB)promoter (Shipley et al., Genetics (1991) 10; 1009-1018), and ubiquitinpromoters such as those isolated from human ubiquitin A, human ubiquitinB, and human ubiquitin C, as described in U.S. Pat. No. 6,667,174,incorporated herein by reference in its entirety. To prolong expression,other regulatory elements may additionally be operably linked to thetransgene, such as, e.g., the Woodchuck Hepatitis Virus Post-RegulatoryElement (WPRE)(Donello et al., J. Virol. (1998) 72:5085-5092) or thebovine growth hormone (BGH) polyadenylation site. In addition, sequencesderived from nonviral genes, such as the murine metallothionein gene,will also find use herein. Such promoter sequences are commerciallyavailable from, e.g., Stratagene (San Diego, Calif.).

For some CNS gene therapy applications, it may be necessary to controltranscriptional activity. To this end, pharmacological regulation ofgene expression with viral vectors can been obtained by includingvarious regulatory elements and drug-responsive promoters as described,for example, in Habermaet al., Gene Ther. (1998) 5.1604-16011; and Ye etal., Science (1995) 283:88-91.

The AAV expression vector which harbors the polynucleotide molecule ofinterest bounded by AAV ITRs, can be constructed by directly insertingthe selected sequence(s) into an AAV genome which has had the major AAVopen reading frames (“ORFs”) excised therefrom. Other portions of theAAV genome can also be deleted, so long as a sufficient portion of theITRs remain to allow for replication and packaging functions. Suchconstructs can be designed using techniques well known in the art. See,e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International PublicationNos. WO 92/01070 (published 23 Jan. 1992) and WO 93/03769 (published 4Mar. 1993); Lebkowski et al. (1988) Molec. Cell. Biol. 1:3988-3996;Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press);Carter (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka(1992) Current Topics in Microbiol. and Immunol. 128:97-129; Kotin(1994) Human Gene Therapy 1:793-801; Shelling and Smith (1994) GeneTherapy 1:165-169; and Zhou et al. (1994) J. Exp. Med 171:1867-1875.

Alternatively, AAV ITRs can be excised from the viral genome or from anAAV vector containing the same and fused 5′ and 3′ of a selected nucleicacid construct that is present in another vector using standard ligationtechniques, such as those described in Sambrook et al., supra. Forexample, ligations can be accomplished in 20 mM Tris-CI pH 7.5, 10 mMMgCl2, 10 mM DTT, 33 μg/ml BSA, 10 mM-50 mM NaCl, and either 40 μM ATP,0.01-0.02 (Weiss) units T4 DNA ligase at 0° C. (for “sticky end”ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14° C.(for “blunt end” ligation). Intermolecular “sticky end” ligations areusually performed at 30-100 μg/ml total DNA concentrations (5-100 nMtotal end concentration). AAV vectors which contain ITRs have beendescribed in, e.g., U.S. Pat. No. 5,139,941. In particular, several AAVvectors are described therein which are available from the American TypeCulture Collection (“ATCC”) under Accession Numbers 53222, 53223, 53224,53225 and 53226.

In certain embodiments, the rAAV expression vectors are provided asself-complementary rAAV constructs. Typically, rAAV DNA is packaged intothe viral capsid as a single-stranded DNA (ssDNA) molecule about 4600nucleotides in length. Following infection of the cell by the virus, thesingle DNA strand is converted into a double-stranded DNA (dsDNA) form.Only the dsDNA is useful to proteins of the cell that transcribe thecontained gene or genes into RNA. Thus, the conventional replicationscheme of AAV requires de novo synthesis of a complementary DNA strand.This step of converting the ssDNA AAV genome into dsDNA prior toexpression can be circumvented by the use of self-complementary (sc)vectors.

Self-complementary vectors are produced by base pairing complementarystrands from two infecting viruses, which does not require DNA synthesis(see, e.g., Nakai et al., J. Virol. (2000) 74:9451-9463). Thisinterstrand base pairing, or strand annealing (SA), is possible becauseAAV packages either the plus or minus DNA strand with equal efficiency(Berns, K. I., Microbiol. Rev. (1990) 54:316-329).

Thus and without being limited as to theory, the need for dsDNAconversion, either by SA or DNA synthesis, can be entirely circumventedby packaging both strands as a single molecule. This can be achieved bytaking advantage of the tendency of AAV to produce dimeric invertedrepeat genomes during the AAV replication cycle. If these dimers aresmall enough, they can be packaged in the same manner as conventionalAAV genomes, and the two halves of the ssDNA molecule can fold and basepair to form a dsDNA molecule of half the length. dsDNA conversion isindependent of host-cell DNA synthesis and vector concentration (McCartyet al., Gene Ther. (2001) 8:1248-1254).

scAAV viral constructs include approximately 4.6 kb and are able to bepackaged into the normal AAV capsid. Each of the known AAV serotypes iscapable of packaging scAAV genomes with similar efficiency (see, e.g.,Sipo et al., Gene Ther. (2007) 14:1319-1329). Thus, in certainembodiments of the instant invention, the scAAV vector comprises capsidproteins from serotypes selected from AAV1, AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, or AAV9 serotypes. However, an scAAV vector maycomprise capsid proteins from any of the known serotypes or modifiedcapsid proteins known in the art. These scAAV vectors may also bepseudotyped vectors comprising which contain the genome of one AAVserotype in the capsid of a second AAV serotype. Such vectors maycomprise, for example, an AAV vector that contains the AAV2 capsid andthe AAV1 genome or an AAV vector that contains the AAV5 capsid and theAAV 2 genome (Auricchio et al., (2001) Hum. Mol. Genet.,10(26):3075-81).

Initially, it was believed that the transgene sequence in an scAAVvector could only comprise approximately 2.2 kb. However, it appearsthere is greater latitude in packaging capacity than previouslybelieved. For example, Wu et al., Human Gene Ther. (2007) 18:171-182successfully packaged scAAV-2 constructs exceeding 3,300 bp anddemonstrated dimeric inverted repeat genomes that were fully DNaseresistant. These vectors yielded the expected increases in transductionefficiency over ssAAV when tested on cultured cells.

scAAV vectors can be produced either by generating vector plasmids thatare approximately half of the conventional genome size combined withselective purification of the infectious double stranded form, orthrough the use of approximately half-genome sized vector plasmids witha mutation in one of the terminal resolution sequences of the AAV virusthat provides for synthesis of double-stranded virus. Both strategiesgenerate + and − strand viral genomes that are covalently linked at oneterminal repeat.

In particular, the generation of normal monomeric AAV genomes relies onthe efficient resolution of the two ITRs in turn, with each round of DNAsynthesis. This reaction is mediated by the ssDNA endonuclease activityof the two larger isoforms of AAV Rep. Nicking the ITR at the terminalresolution site is followed by DNA elongation from the nick by host DNApolymerase. Dimeric genomes are formed when Rep fails to nick theterminal resolution site before it is reached by the replication complexinitiated at the other end.

The yield of dimeric genomes in a scAAV prep can be increaseddramatically by inhibiting resolution at one terminal repeat. This isreadily accomplished by deleting the terminal resolution site sequencefrom one ITR, such that the Rep protein cannot generate the essentialssDNA nick (see, e.g., McCarty et al., Gene Ther. (2003) 10:2112-2118and Wang et al., Gene Ther. (2003) 10:2105-2111). The replicationcomplex initiated at the other ITR then copies through the hairpin andback toward the initiating end. Replication proceeds to the end of thetemplate molecule, leaving a dsDNA inverted repeat with a wild-type ITRat each end and the mutated ITR in the middle. This dimeric invertedrepeat can then undergo normal rounds of replication from the twowild-type ITR ends. Each displaced daughter strand comprises a ssDNAinverted repeat with a complete ITR at each end and a mutated ITR in themiddle. Packaging into the AAV capsid starts at the 3′ end of thedisplaced strand. Production of scAAV from constructs with one mutatedITR typically yields more than 90% dimeric genomes.

Production and purification of scAAV vector from mutated ITR constructsis the same as conventional ssAAV, as described further below. However,if dot blot or Southern blot is used, the vector DNA is preferablyapplied to hybridization membranes under alkaline conditions to preventreannealing of the complementary strands. Additionally, it is possiblefor a spurious Rep-nicking site to be produced close enough to themutated ITR to allow terminal resolution and generation of monomergenomes. This can typically be avoided by turning the transgene cassettearound with respect to the mutant and wild-type terminal repeats.

See, e.g., McCarty, D. M., Molec. Ther. (2008) 16:1648-1656; McCarty etal., Gene Ther. (2001) 8:1248-1254; McCarty et al., Gene Ther. (2003)10:2112-2118; Wang et al., Gene Ther. (2003) 10:2105-2111); Wu et al.,Human Gene Ther. (2007) 18:171-182; U.S. Patent Publication Nos.2007/0243168 and 2007/0253936, incorporated herein by reference in theirentireties; as well as the examples herein, for methods of producingscAAV constructs.

For the purposes of the invention, suitable host cells for producingrAAV virions from the AAV expression vectors (either conventional or scvectors) include microorganisms, yeast cells, insect cells, andmammalian cells, that can be, or have been, used as recipients of aheterologous DNA molecule and that are capable of growth in, forexample, suspension culture, a bioreactor, or the like. The termincludes the progeny of the original cell which has been transfected.Thus, a “host cell” as used herein generally refers to a cell which hasbeen transfected with an exogenous DNA sequence. Cells from the stablehuman cell line, 293 (readily available through, e.g., the American TypeCulture Collection under Accession Number ATCC CRL1573) are preferred inthe practice of the present invention. Particularly, the human cell line293 is a human embryonic kidney cell line that has been transformed withadenovirus type-S DNA fragments (Graham et al. (1977) J. Gen. Viol.36:59), and expresses the adenoviral Eta and E1b genes (Aiello et al.(1979) Virology 94:460). The 293 cell line is readily transfected, andprovides a particularly convenient platform in which to produce rAAVvirions.

AAV Helper Functions

Host cells containing the above-described AAV expression vectors must berendered capable of providing AAV helper functions in order to replicateand encapsidate the nucleotide sequences flanked by the AAV ITRs toproduce rAAV virions. AAV helper functions are generally AAV-derivedcoding sequences which can be expressed to provide AAV gene productsthat, in turn, function in trans for productive AAV replication. AAVhelper functions are used herein to complement necessary AAV functionsthat are missing from the AAV expression vectors. Thus, AAV helperfunctions include one, or both of the major AAV ORFs, namely the rep andcap coding regions, or functional homologues thereof.

By “AAV rep coding region” is meant the art-recognized region of the AAVgenome which encodes the replication proteins Rep 78, Rep 68, Rep 52 andRep 40. These Rep expression products have been shown to possess manyfunctions, including recognition, binding and nicking of the AAV originof DNA replication, DNA helicase activity and modulation oftranscription from AAV (or other heterologous) promoters. The Repexpression products are collectively required for replicating the AAVgenome. For a description of the AAV rep coding region, see, e.g.,Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol.118:97-129; and Kotin, R. M. (1994) Human Gene Therapy 5:793-801.Suitable homologues of the AAV rep coding region include the humanherpesvirus 6 (HHV-6) rep gene which is also known to mediate AAV-2 DNAreplication (Thomson et al. (1994) Virology 204:304-311).

By “AAV cap coding region” is meant the art-recognized region of the AAVgenome which encodes the capsid proteins VPI, VP2, and VP3, orfunctional homologues thereof. These Cap expression products supply thepackaging functions which are collectively required for packaging theviral genome. For a description of the AAV cap coding region, see, e.g.,Muzyczka, N. and Kotin, R. M. (supra).

AAV helper functions are introduced into the host cell by transfectingthe host cell with an AAV helper construct either prior to, orconcurrently with, the transfection of the AAV expression vector. AAVhelper constructs are thus used to provide at least transient expressionof AAV rep and/or cap genes to complement missing AAV functions that arenecessary for productive AAV infection. AAV helper constructs lack AAVITRs and can neither replicate nor package themselves.

These constructs can be in the form of a plasmid, phage, transposon,cosmid, virus, or virion. A number of AAV helper constructs have beendescribed, such as the commonly used plasmids pAAV/Ad and pIM29+45 whichencode both Rep and Cap expression products. See, e.g., Samulski et al.(1989) J. Virol. 63:3822-3828; and McCarty et al. (1991) J. Virol.65:2936-2945. A number of other vectors have been described which encodeRep and/or Cap expression products. See, e.g., U.S. Pat. No. 5,139,941.

AAV Accessory Functions

The host cell (or packaging cell) must also be rendered capable ofproviding nonAAV-derived functions, or “accessory functions,” in orderto produce rAAV virions. Accessory functions are nonAAV-derived viraland/or cellular functions upon which AAV is dependent for itsreplication. Thus, accessory functions include at least those nonAAVproteins and RNAs that are required in AAV replication, including thoseinvolved in activation of AAV gene transcription, stage specific AAVmRNA splicing, AAV DNA replication, synthesis of Cap expression productsand AAV capsid assembly. Viral-based accessory functions can be derivedfrom any of the known helper viruses.

In particular, accessory functions can be introduced into and thenexpressed in host cells using methods known to those of skill in theart. Typically, accessory functions are provided by infection of thehost cells with an unrelated helper virus. A number of suitable helperviruses are known, including adenoviruses; herpesviruses such as herpessimplex virus types 1 and 2; and vaccinia viruses. Nonviral accessoryfunctions will also find use herein, such as those provided by cellsynchronization using any of various known agents. See, e.g., Buller etal. (1981) J. Virol. Q:241-247; McPherson et al. (1985) Virology14.7:217-222; Schlehofer et al. (1986) Virology 152:110-117.

Alternatively, accessory functions can be provided using an accessoryfunction vector as defined above. See, e.g., U.S. Pat. No. 6,004,797 andInternational Publication No. WO 01/83797, incorporated herein byreference in their entireties. Nucleic acid sequences providing theaccessory functions can be obtained from natural sources, such as fromthe genome of an adenovirus particle, or constructed using recombinantor synthetic methods known in the art. As explained above, it has beendemonstrated that the full-complement of adenovirus genes are notrequired for accessory helper functions. In particular, adenovirusmutants incapable of DNA replication and late gene synthesis have beenshown to be permissive for AAV replication. Ito et al., (1970) J. Gen.Virol. 9:243; Ishibashi et al, (1971) Virology 45:317. Similarly,mutants within the E2B and E3 regions have been shown to support AAVreplication, indicating that the E2B and E3 regions are probably notinvolved in providing accessory functions. Carter et al., (1983)Virology 126:505. However, adenoviruses defective in the E1 region, orhaving a deleted E4 region, are unable to support AAV replication. Thus,E1A and E4 regions are likely required for AAV replication, eitherdirectly or indirectly. Laughlin et al., (1982) J. Virol. 41.:868; Janiket al., (1981) Proc. Natl. Acad. Sci. USA 78:1925; Carter et al., (1983)Virology 126:505. Other characterized Ad mutants include: E1B (Laughlinet al. (1982), supra; Janik et al. (1981), supra; Ostrove et al., (1980)Virology 104:502); E2A (Handa et al., (1975) J. Gen.Virol. 22:239;Strauss et al., (1976) J. Virol. 0.12:140; Myers et al., (1980) J.Virol. 11:665; Jay et al., (1981) Proc. Natl. Acad. Sci. USA 21:2927;Myers et al., (1981) J. Biol. Chem. 256:567); E2B (Carter,Adeno-Associated Virus Helper Functions, in I CRC Handbook ofParvoviruses (P. Tijssen ed., 1990)); E3 (Carter et al. (1983), supra);and E4 (Carter et al. (1983), supra; Carter (1995)). Although studies ofthe accessory functions provided by adenoviruses having mutations in theE1B coding region have produced conflicting results, Samulski et al.,(1988) J. Virol. 62:206-210, has reported that E1B55k is required forAAV virion production, while E1B19k is not. In addition, InternationalPublication WO 97/17458 and Matshushita et al., (1998) Gene Therapy5:938-945, describe accessory function vectors encoding various Adgenes. Particularly preferred accessory function vectors comprise anadenovirus VA RNA coding region, an adenovirus E4 ORF6 coding region, anadenovirus E2A 72 kD coding region, an adenovirus E1A coding region, andan adenovirus Et B region lacking an intact E1B55k coding region. Suchvectors are described in International Publication No. WO 01/83797.

As a consequence of the infection of the host cell with a helper virus,or transfection of the host cell with an accessory function vector,accessory functions are expressed which transactivate the AAV helperconstruct to produce AAV Rep and/or Cap proteins. The Rep expressionproducts excise the recombinant DNA (including the DNA of interest) fromthe AAV expression vector. The Rep proteins also serve to duplicate theAAV genome. The expressed Cap proteins assemble into capsids, and therecombinant AAV genome is packaged into the capsids. Thus, productiveAAV replication ensues, and the DNA is packaged into rAAV virions. A“recombinant AAV virion,” or “rAAV virion” is defined herein as aninfectious, replication-defective virus including an AAV protein shell,encapsidating a heterologous nucleotide sequence of interest which isflanked on both sides by AAV ITRs.

Following recombinant AAV replication, rAAV virions can be purified fromthe host cell using a variety of conventional purification methods, suchas column chromatography, CsCl gradients, and the like. For example, aplurality of column purification steps can be used, such as purificationover an anion exchange column, an affinity column and/or a cationexchange column. See, for example, International Publication No. WO02/12455. Further, if infection is employed to express the accessoryfunctions, residual helper virus can be inactivated, using knownmethods. For example, adenovirus can be inactivated by heating totemperatures of approximately 60° C. for, e.g., 20 minutes or more. Thistreatment effectively inactivates only the helper virus since AAV isextremely heat stable while the helper adenovirus is heat labile.

The resulting rAAV virions containing the nucleotide sequence ofinterest can then be used for gene delivery using the techniquesdescribed below.

Compositions and Delivery

A. Compositions

Once produced, the rAAV virions encoding the gene of interest, will beformulated into compositions suitable for delivery. Compositions willcomprise sufficient genetic material to produce a therapeuticallyeffective amount of the gene of interest, i.e., an amount sufficient to(1) prevent the development of the disease or cause the disease to occurwith less intensity in a subject that may be exposed to or predisposedto the disease but does not yet experience or display symptoms of thedisease, (2) inhibit the disease, i.e., arrest the development orreverse the disease state, or (3) relieve symptoms of the disease i.e.,decrease the number of symptoms experienced by the subject, as well aschange the cellular pathology associated with the disease.

Appropriate doses will also depend on the mammal being treated (e.g.,human or nonhuman primate or other mammal), age and general condition ofthe subject to be treated, the severity of the condition being treated,the mode of administration, among other factors. An appropriateeffective amount can be readily determined by one of skill in the artand representative amounts are provided below.

The compositions will also contain a pharmaceutically acceptableexcipient. Such excipients include any pharmaceutical agent that doesnot itself induce the production of antibodies harmful to the individualreceiving the composition, and which may be administered without unduetoxicity. Pharmaceutically acceptable excipients include, but are notlimited to, sorbitol, any of the various TWEEN compounds, and liquidssuch as water, saline, glycerol and ethanol. Pharmaceutically acceptablesalts can be included therein, for example, mineral acid salts such ashydrochlorides, hydrobromides, phosphates, sulfates, and the like; andthe salts of organic acids such as acetates, propionates, malonates,benzoates, and the like. Additionally, auxiliary substances, such aswetting or emulsifying agents, pH buffering substances, and the like,may be present in such vehicles. A thorough discussion ofpharmaceutically acceptable excipients is available in REMINGTON'SPHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991).

Formulations can be liquid or solid, for example, lyophilized.Formulations can also be administered as aerosols.

One particularly useful formulation comprises the rAAV virion ofinterest in combination with one or more dihydric or polyhydricalcohols, and, optionally, a detergent, such as a sorbitan ester. See,for example, U.S. Pat. No. 6,764,845, incorporated herein by referencein its entirety.

B. Delivery

Generally, the recombinant virions are introduced into the subject usingeither in vivo or in vitro transduction techniques. If transduced invitro, the desired recipient cell will be removed from the subject,transduced with the recombinant vector and reintroduced into thesubject. Alternatively, syngeneic or xenogeneic cells can be used wherethose cells will not generate an inappropriate immune response in thesubject. Suitable cells for delivery to mammalian host animals includemammalian cell types from organs, tumors, or cell lines. For example,human, murine, goat, ovine, bovine, dog, cat, and porcine cells can beused. Suitable cell types for use include without limitation,fibroblasts, hepatocytes, endothelial cells, keratinocytes,hematopoietic cells, epithelial cells, myocytes, neuronal cells, andstem cells. Additionally, neural progenitor cells can be transduced invitro and then delivered to the CNS.

Cells can be transduced in vitro by combining recombinant virions withthe desired cell in appropriate media, and the cells can be screened forthose cells harboring the DNA of interest using conventional techniquessuch as Southern blots and/or PCR, or by using selectable markers.Transduced cells can then be formulated into pharmaceuticalcompositions, as described above, and the composition introduced intothe subject by various techniques as described below, in one or moredoses.

For in vivo delivery, the recombinant virions will be formulated intopharmaceutical compositions and one or more dosages may be administereddirectly in the indicated manner. For identification of structures inthe human brain, see, e.g., The Human Brain: Surface, Three-DimensionalSectional Anatomy With MRI, and Blood Supply, 2nd ed., eds. Deuteron etal., Springer Vela, 1999; Atlas of the Human Brain, eds. Mai et al.,Academic Press; 1997; and Co-Planar Sterotaxic Atlas of the Human Brain:3-Dimensional Proportional System: An Approach to Cerebral Imaging, eds.Tamarack et al., Thyme Medical Pub., 1988. For identification ofstructures in the mouse brain, see, e.g., The Mouse Brain in SterotaxicCoordinates, 2nd ed., Academic Press, 2000. If desired, the human brainstructure can be correlated to similar structures in the brain ofanother mammal. For example, most mammals, including humans and rodents,show a similar topographical organization of the entorhinal-hippocampusprojections, with neurons in the lateral part of both the lateral andmedial entorhinal cortex projecting to the dorsal part or septal pole ofthe hippocampus, whereas the projection to the ventral hippocampusoriginates primarily from neurons in medial parts of the entorhinalcortex (Principles of Neural Science, 4th ed., eds Kandel et al.,McGraw-Hill, 1991; The Rat Nervous System, 2nd ed., ed. Paxinos,Academic Press, 1995). Furthermore, layer II cells of the entorhinalcortex project to the dentate gyrus, and they terminate in the outertwo-thirds of the molecular layer of the dentate gyrus. The axons fromlayer III cells project bilaterally to the cornu ammonis areas CA1 andCA3 of the hippocampus, terminating in the stratum lacunose molecularlayer.

To deliver the vector specifically to a particular region of the centralnervous system, especially to a particular region of the brain, it maybe administered by sterotaxic microinjection. For example, on the day ofsurgery, patients will have the sterotaxic frame base fixed in place(screwed into the skull). The brain with sterotaxic frame base(MRI-compatible with fiduciary markings) will be imaged using highresolution MRI. The MRI images will then be transferred to a computerthat runs stereotaxic software. A series of coronal, sagittal and axialimages will be used to determine the target site of vector injection,and trajectory. The software directly translates the trajectory into3-dimensional coordinates appropriate for the stereotaxic frame. Burrholes are drilled above the entry site and the stereotaxic apparatuslocalized with the needle implanted at the given depth. The vector in apharmaceutically acceptable carrier will then be injected. The vector isthen administrated by direct injection to the primary target site andretrogradely transported to distal target sites via axons. Additionalroutes of administration may be used, e.g., superficial corticalapplication under direct visualization, or other non-stereotaxicapplication.

Recombinant AAV of any serotype can be used in the instant invention,wherein the recombinant AAV may be either a self-complementary AAV or anon-self complementary AAV. The serotype of the viral vector used incertain embodiments of the invention is selected from the groupconsisting from AAV 1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, andAAV9 (see, e.g., Gao et al. (2002) PNAS, 99:11854 11859; and ViralVectors for Gene Therapy: Methods and Protocols, ed. Machida, HumanaPress, 2003). Other serotype besides those listed herein can be used.Furthermore, pseudotyped AAV vectors may also be utilized in the methodsdescribed herein. Pseudotyped AAV vectors are those which contain thegenome of one AAV serotype in the capsid of a second AAV serotype; forexample, an AAV vector that contains the AAV2 capsid and the AAV1 genomeor an AAV vector that contains the AAV5 capsid and the AAV 2 genome(Auricchio et al., (2001) Hum. Mol. Genet., 10(26):3075-81).

Recombinant virions or cells transduced in vitro may be delivereddirectly to neural tissue such as peripheral nerves, the retina, dorsalroot ganglia, neuromuscular junction, as well as the CNS, by injectioninto, e.g., the ventricular region, such as one or both of the lateralventricles, as well as to the striatum (e.g., the caudate nucleus orputamen of the striatum), the cerebellum, spinal cord, and neuromuscularjunction, with a needle, catheter or related device, using neurosurgicaltechniques known in the art, such as by stereotactic injection (see,e.g., Stein et al., J Virol 73:3424-3429, 1999; Davidson et al., PNAS97:3428-3432, 2000; Davidson et al., Nat.Genet. 3:219-223, 1993; andAlisky and Davidson, Hum. Gene Ther. 11:2315-2329, 2000). In anillustrative embodiment, the delivery is accomplished by directinjection of a high titer vector solution into the spinal cord of asubject or patient.

In another illustrative embodiment, a method to deliver a transgene tothe spinal cord and/or the brainstem region of a subject byadministering a recombinant AAV vector containing the transgene to atleast one region of the deep cerebellar nuclei (DCN) region of thecerebellum of the subject's brain. Deep within the cerebellum is greymatter called the deep cerebellar nuclei termed the medial (fastigial)nucleus, the interposed (interpositus) nucleus and the lateral (dentate)nucleus. As used herein, the term “deep cerebellar nuclei” collectivelyrefers to these three regions, wherein one or more of these threeregions may be targeted. The viral delivery is under conditions thatfavor expression of the transgene in the spinal cord and/or thebrainstem region. in at least one subdivision of the spinal cord of thesubject. These subdivisions include one or more of cervical, thoracic,lumbar or sacral.

Without being limited as to theory, one embodiment of the invention liesin the ability to provide a therapeutic molecule (for example, a proteinor peptide) to each division of the spinal cord. This may beaccomplished by injecting an AAV vector, including a scAAV vector intothe DCN. Furthermore, it may be important to target individual laminawithin each spinal cord division. Lamina are specific sub-regions withinregions of the brain and spinal cord. It may be desirable in certainembodiments to target specific lamina within a certain spinal corddivision. Since motor neuron damage may occur within the upper motorneurons as well, it may also be desirable to provide a therapeuticmolecule (for example, a protein or peptide) to the divisions of thebrainstem. In one embodiment, it may be desirable to provide thetherapeutic molecule to both the spinal cord, including some or allsubdivisions as well as to the brainstem, including some or allsubdivisions. The instant invention uses the introduction of an AAVvector into the DCN to accomplish the above described delivery of atherapeutic molecule to the spinal cord region(s) and/or brainstem.

Another method for targeting spinal cord (e.g., glia) is by intrathecaldelivery, rather than into the spinal cord tissue itself. Such deliverypresents many advantages. The targeted protein is released into thesurrounding CSF and unlike viruses, released proteins can penetrate intothe spinal cord parenchyma, just as they do after acute intrathecalinjections. Indeed, intrathecal delivery of viral vectors can keepexpression local. An additional advantage of intrathecal gene therapy isthat the intrathecal route mimics lumbar puncture administration (i.e.,spinal tap) already in routine use in humans.

Another method for administering the recombinant vectors or transducedcells is by delivery to dorsal root ganglia (DRG) neurons, e.g., byinjection into the epidural space with subsequent diffusion to DRG. Forexample, the recombinant vectors or transduced cells can be deliveredvia intrathecal cannulation under conditions where the protein isdiffused to DRG. See, e.g., Chiang et al., Acta Anaesthesiol. Sin (2000)38:31-36; Jain, K. K., Expert Opin. Investig. Drugs (2000) 9:2403-2410.

Yet another mode of administration to the CNS uses a convection-enhanceddelivery (CED) system, which is any non-manual delivery of the vector.In one embodiment of CED, a pressure gradient is created via the use ofa non-manual delivery system. By using CED, recombinant vectors can bedelivered to many cells over large areas of the CNS. Moreover, thedelivered vectors efficiently express transgenes in CNS cells (e.g.,glial cells). Any convection-enhanced delivery device may be appropriatefor delivery of recombinant vectors. In a preferred embodiment, thedevice is an osmotic pump or an infusion pump. Both osmotic and infusionpumps are commercially available from a variety of suppliers, forexample Alzet Corporation, Hamilton Corporation, Alza, Inc., Palo Alto,Calif.). Typically, a recombinant vector is delivered via CED devices asfollows. A catheter, cannula or other injection device is inserted intoCNS tissue in the chosen subject. Stereotactic maps and positioningdevices are available, for example from ASI Instruments, Warren, Mich.Positioning may also be conducted by using anatomical maps obtained byCT and/or MRI imaging to help guide the injection device to the chosentarget. Moreover, because the methods described herein can be practicedsuch that relatively large areas of the subject take up the recombinantvectors, fewer infusion cannula are needed. Since surgical complicationsare often related to the number of penetrations, this mode of deliveryserves to reduce the side-effects seen with conventional deliverytechniques. For a detailed description regarding CED delivery, see U.S.Pat. No. 6,309,634, incorporated herein by reference in its entirety.

Intracerebroventricular, or intraventricular, delivery of a recombinantAAV vector may be performed in any one or more of the brain'sventricles, which are filled with cerebrospinal fluid (CSF). CSF is aclear fluid that fills the ventricles, is present in the subarachnoidspace, and surrounds the brain and spinal cord. CSF is produced by thechoroid plexuses and via the weeping or transmission of tissue fluid bythe brain into the ventricles. The choroid plexus is a structure liningthe floor of the lateral ventricle and the roof of the third and fourthventricles. Certain studies have indicated that these structures arecapable of producing 400-600 ccs of fluid per day consistent with anamount to fill the central nervous system spaces four times in a day. Inadult humans, the volume of this fluid has been calculated to be from125 to 150 ml (4-5 oz). The CSF is in continuous formation, circulationand absorption. Certain studies have indicated that approximately 430 to450 ml (nearly 2 cups) of CSF may be produced every day. Certaincalculations estimate that production equals approximately 0.35 ml perminute in adults and 0.15 per minute in infant humans. The choroidplexuses of the lateral ventricles produce the majority of CSF. It flowsthrough the foramina of Monro into the third ventricle where it is addedto by production from the third ventricle and continues down through theaqueduct of Sylvius to the fourth ventricle. The fourth ventricle addsmore CSF; the fluid then travels into the subarachnoid space through theforamina of Magendie and Luschka. It then circulates throughout the baseof the brain, down around the spinal cord and upward over the cerebralhemispheres. The CSF empties into the blood via the arachnoid villi andintracranial vascular sinuses.

In one aspect, the disclosed methods include administering to the CNS ofan afflicted subject a rAAV virion carrying a transgene encoding atherapeutic product and allowing the transgene to be expressed withinthe CNS near the administration site at a level sufficient to exert atherapeutic effect as the expressed protein is transported via the CSFthroughout the CNS. In some embodiments, the methods compriseadministration of a high titer virion composition carrying a therapeutictransgene so that the transgene product is expressed at a therapeuticlevel at a first site within the CNS distal to the ultimate site ofaction of the expressed product.

In experimental mice, the total volume of injected AAV solution is forexample, between 1 to 20 μl. For other mammals, including the human,volumes and delivery rates are appropriately scaled. Treatment mayconsist of a single injection per target site, or may be repeated in oneor more sites. Multiple injection sites can be used. For example, insome embodiments, in addition to the first administration site, acomposition containing a viral vector carrying a transgene isadministered to another site which can be contralateral or ipsilateralto the first administration site. Injections can be single or multiple,unilateral or bilateral.

Dosage treatment may be a single dose schedule, continuously orintermittently, or a multiple dose schedule. Moreover, the subject maybe administered as many doses as appropriate. If multiple doses areadministered, the first formulation administered can be the same ordifferent than the subsequent formulations. Thus, for example, the firstadministration can be in the form of an AAV vector and the secondadministration in the form of an adenovirus vector, plasmid DNA, aprotein composition, or the like. Moreover, subsequent delivery can alsobe the same or different than the second mode of delivery.

In addition, the subject may receive the rAAV vector of the instantinvention by a combination of the delivery methods disclosed therein.Thus, a subject may receive injections of an AAV vector in at least twoinjection sites selected from the group consisting ofintracerebroventricular injections, direct spinal cord injections,intrathecal injections, and intraparenchymal brain injections (e.g., thestriatum, the cerebellum, including the deep cerebellar nuclei). In oneembodiment, the subject may receive rAAV vector via 1) at least oneintracerebroventricular injection, and at least one direct spinal cordinjection or 2) at least one intracerebroventricular injection and atleast one intrathecal injection or 3) at least oneintracerebroventricular injection and at least one intraparenchymalbrain injection or 4) at least one direct spinal cord injection and atleast one intrathecal injection or 5) at least one direct spinal cordinjection and at least one intraparenchymal brain injection or 6) atleast one intrathecal injection and at least one intraparenchymal braininjection.

It should be understood that more than one transgene can be expressed bythe delivered recombinant virion. Alternatively, separate vectors, eachexpressing one or more different transgenes, can also be delivered tothe subject as described herein. Thus, multiple transgenes can bedelivered concurrently or sequentially. Furthermore, it is also intendedthat the vectors delivered by the methods of the present invention becombined with other suitable compositions and therapies. Additionally,combinations of protein and nucleic acid treatments can be used.

Methods of determining the most effective means of administration andtherapeutically effective dosages are well known to those of skill inthe art and will vary with the vector, the composition of the therapy,the target cells, and the subject being treated. Therapeuticallyeffective doses can be readily determined using, for example, one ormore animal models of the particular disease in question. A“therapeutically effective amount” will fall in a relatively broad rangethat can be determined through clinical trials. For example, for in vivoinjection of rAAV virions, a dose will be on the order of from about 10⁶to 10¹⁵ genome particles of the recombinant virus, more preferably 10⁸to 10¹⁴ genome particles recombinant virus, or any dose within theseranges which is sufficient to provide the desired affect. In certainembodiments, the concentration or titer of the vector in the compositionis at least: (a) 5 6, 7, 8, 9, 10, 15, 20, 25, or 50 (x10¹² gp/ml); (b)5 6, 7, 8, 9, 10, 15, 20, 25, or 50 (x10⁹ tu/ml); or (c) 5, 6, 7, 8, 9,10, 15, 20, 25, or 50 (x10¹⁰ iu/ml).

For in vitro transduction, an effective amount of rAAV virions to bedelivered to cells will be on the order of 10⁸ to 10¹³ of therecombinant virus. The amount of transduced cells in the pharmaceuticalcompositions, in turn will be from about 104 to 10¹⁰ cells, morepreferably 10⁵ to 10⁸ cells. Other effective dosages can be readilyestablished by one of ordinary skill in the art through routine trialsestablishing dose response curves.

Generally, from 1 μl to 1 ml of composition will be delivered, such asfrom 0.01 to about 0.5 ml, for example about 0.05 to about 0.3 ml, suchas 0.08, 0.09, 0.1, 0.2, etc. and any number within these ranges, ofcomposition will be delivered.

Animal Models

Therapeutic effectiveness and safety using the AAV virions includingtransgenes as described above can be tested in an appropriate animalmodel. For example, animal models which appear most similar to humandisease include animal species which either spontaneously develop a highincidence of the particular disease or those that have been induced todo so.

In particular, several animal models for SMA are known and have beengenerated. See, e.g., Sumner C. J., NeuroRx (2006) 2:235-245; Schmid etal., J. Child Neurol. (2007) 22:1004-1012. As explained above, themolecular basis of SMA, an autosomal recessive neuromuscular disorder,is the homozygous loss of the survival motor neuron gene 1 (SMN1). Anearly identical copy of the SMN1 gene, called SMN2 is found in humansand modulates the disease severity. In contrast to humans, mice have asingle gene (SMN) that is equivalent to SMN1. Homozygous loss of thisgene is lethal to embryos and results in massive cell death, whichindicates that the SMN gene product is necessary for cellular survivaland function. The introduction of 2 copies of SMN2 into mice lacking SMNrescues the embryonic lethality, resulting in mice with the SMAphenotype (Monani et al., Hum. Mol. Genet. (2000) 2:333-339. A high copynumber of SMN2 rescues the mice because sufficient SMN protein isproduced in motor neurons. See, also, Hsieh-Li, et al., Nat. Genet.(2000) 2:66-70, reporting the production of transgenic mouse lines thatexpressed human SMN2. In particular, transgenic mice harboring SMN2 inthe SMN−/− background show pathological changes in the spinal cord andskeletal muscles similar to those of SMA patients. The severity of thepathological changes in these mice correlates with the amount of SMNprotein that contained the region encoded by exon 7. Phenotypes in thismouse model include motor neuron cell loss, skeletal muscle atrophy,aberrant neuromuscular junctions (NMJ), behavioral deficits, paralysis,and a shortened life span of about two weeks. Le et al., Hum. Mol.Genet. (2005) 14:845-857.

Similarly, animal models for ALS are known. ALS is a fatalneurodegenerative disease that is characterized by a selective loss ofmotor neurons in the cortex, brain stem and spinal cord. Progression ofthe disease can lead to atrophy of limb, axial and respiratory muscles.Motor neuron cell death is accompanied by reactive gliosis,neurofilament abnormalities, and a significant loss of large myelinatedfibers in the corticospinal tracts and ventral roots. Although theetiology of ALS is poorly understood, accumulating evidence indicatesthat sporadic (SALS) and familial (FALS) ALS share many similarpathological features; thus, providing a hope that the study of eitherform will lead to a common treatment. FALS accounts for approximately10% of diagnosed cases, of which 20% are associated with dominantlyinherited mutations in Cu/Zn superoxide dismutase (SODI). Transgenicmice that express the mutant human SODI protein (e.g., SODIG93A mice)recapitulate many pathological features of ALS and are an availableanima model to study ALS. For SALS, a myriad of pathological mechanismshave been implicated as the underlying cause, including glutamateinduced excitotoxicity, toxin exposure, proteasome dysfunction,mitochondrial damage, neurofilament disorganization and loss ofneurotrophic support.

Experimental Autoimmune Encephalomyelitis (EAE), also calledExperimental Allergic Encephalomyelitis, provides an animal model forMS. EAE resembles the various forms and stages of MS very closely. EAEis an acute or chronic-relapsing, acquired, inflammatory anddemyelinating autoimmune disease. In order to create the disease,animals are injected with proteins that make up myelin, the insulatingsheath that surrounds neurons. These proteins induce an autoimmuneresponse in the injected animals which develop a disease process thatclosely resembles MS in humans. EAE has been induced in a number ofdifferent animal species including mice, rats, guinea pigs, rabbits,macaques, rhesus monkeys and marmosets.

Spinal and bulbar muscular atrophy (SBMA) is an adult-onset motor neurondisease, caused by the expansion of a trinucleotide repeat (TNR) in exon1 of the androgen receptor (AR) gene. This disorder is characterized bydegeneration of motor and sensory neurons, proximal muscular atrophy,and endocrine abnormalities, such as gynecomastia and reduced fertility.Only males develop symptoms, while female carriers usually areasymptomatic. The molecular basis of SBMA is the expansion of atrinucleotide CAG repeat, which encodes the polyglutamine (polyQ) tract,in the first exon of the androgen receptor (AR) gene. The pathologichallmark is nuclear inclusions (NIs) containing the mutant and truncatedAR with expanded polyQ in the residual motor neurons in the brainstemand spinal cord as well as in some other visceral organs. Severaltransgenic mouse models have been created for studying the pathogenesisof SBMA. See, e.g., Katsuno et al., Cytogen. and Genome Res. (2003)100:243-251. For example, a transgenic mouse model carrying pure 239CAGs under human AR promoter and another model carrying truncated ARwith expanded CAGs show motor impairment and nuclear Nis in spinal motorneurons. Transgenic mice carrying full-length human AR with expandedpolyQ demonstrate progressive motor impairment and neurogenic pathologyas well as sexual difference of phenotypes. These models recapitulatethe phenotypic expression observed in SBMA.

Machado-Joseph disease (MJD), also called spinocerebellar ataxia type 3,is caused by mutant ataxin-3 with a polyglutamine expansion. Mousemodels of MJD, as well as other polyglutamine spinocerebellar ataxiashave been generated. For a review of these models, see e.g., Gould, V.F. C. NeuroRX(2005) 2:480-483.

Accordingly, animal models standard in the art are available for thescreening and/or assessment for activity and/or effectiveness of themethods and compositions of the invention for the treatment of motorneuron disorders.

Kits of the Invention

The invention also provides kits. In certain embodiments, the kits ofthe invention comprise one or more containers comprising recombinantvectors encoding the protein of interest. The kits may further comprisea suitable set of instructions, generally written instructions, relatingto the use of the vectors for any of the methods described herein.

The kits may comprise the components in any convenient, appropriatepackaging. For example, if the recombinant vectors are provided as a dryformulation (e.g., freeze dried or a dry powder), a vial with aresilient stopper is normally used, so that the vectors may be easilyresuspended by injecting fluid through the resilient stopper. Ampuleswith non-resilient, removable closures (e.g., sealed glass) or resilientstoppers are most conveniently used for liquid formulations. Alsocontemplated are packages for use in combination with a specific device,such as a syringe or an infusion device such as a minipump.

The instructions relating to the use or the recombinant vectorsgenerally include information as to dosage, dosing schedule, and routeof administration for the intended method of use. The containers may beunit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses.Instructions supplied in the kits of the invention are typically writteninstructions on a label or package insert (e.g., a paper sheet includedin the kit), but machine-readable instructions (e.g., instructionscarried on a magnetic or optical storage disk) are also acceptable.

2. Experimental

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

Materials and Methods

AAV vectors. The open reading frame of a exemplary human SMN1 gene (theopen reading frame sequence is shown in FIG. 9A; the corresponding aminoacid sequence is shown in FIG. 9B; the complete nucleotide sequence isfound at GenBank accession number NM_000344)) was cloned into a shuttleplasmid containing either the AAV2 inverted terminal repeats (ITR) andthe 1.6 kb cytomegalovirus enhancer/chicken β-actin (CBA) promoter orthe scAAV2 ITR and the 0.4 kb human β-glucuronidase (GUSB) promoter. Thesize constraint of the recombinant genome in the scAAV packagingreaction required the use of a small promoter (McCarty, D. M. Molec.Ther. (2008) 1k:1648-1656). Thus, the 0.4 kb GUSB promoter was chosenbecause it is ubiquitously expressed throughout the CNS including motorneurons of the spinal cord (Passini et al., J. Virol. (2001)75:12382-12392). The recombinant plasmids were each packaged into AAVserotype-8 capsid by triple-plasmid cotransfection of human 293 cells(see, e.g., U.S. Pat. No. 6,001,650, incorporated by reference herein inits entirety) and virions were column-purified as reported previously(O'Riordan et al., J. Gene Med. (2000) 2:444-454.). The resultingvectors AAV2/8-CBA-hSMN1 (AAV-hSMN1) and scAAV2/8-GUSB-hSMN1(scAAV-hSMN1) possessed titers of 8.3 e12 and 2.8 e12 genome copies perml, respectively.

Animals and procedures. Heterozygote (SMN^(+/−), hSMN2^(+/+),SMNΔ7^(+/+)) breeding pairs were mated and, on the day of birth (P0),newborn pups received 3 total injections of 2 μl each into the cerebrallateral ventricles of both hemispheres and the upper lumbar spinal cord.The total doses of viral vectors were 5.0 e10 and 1.7 e10 genome copiesfor AAV-hSMN1 and scAAV-hSMN1, respectively. All the injections wereperformed with a finely drawn glass micropipette needle as described(Passini et al, J. Virol. (2001) 75:12382-12392). Following theinjections, the pups were toe-clipped and genotyped (Le et al., Hum.Mol. Genet. (2005) 14:845-857) to identify SMA (SMN^(−/−), hSMN2^(+/+),SMNΔ7^(+/+)), heterozygote, and wild type (SMN^(+/+), hSMN2^(+/+),SMNΔ7^(+/+)) mice. All the litters were culled to 7 pups to control forlitter size on survival. Some of the litters were not injected in orderto generate untreated control groups.

Western blots. For biochemical analysis, treated and untreated mice werekilled at 16 and 58-66 days were perfused with phosphate-buffered saline(PBS), the spinal cords were dissected and separated into the lumbar,thoracic and cervical segments, and snap-frozen in liquid nitrogen.Tissues were homogenized at a concentration of 50 mg/mL using T-Perlysis buffer and protease inhibitor cocktail (Pierce, Rockford, Ill.).The homogenates were cleared by centrifugation at 10,000 RCF for 6minutes and the protein concentration was measured by BCA assay (Pierce,Rockford, Ill.). 10-20 μg of homogenate protein were resolved on a 4-12%SDS-PAGE, transferred to nitrocellulose membrane, and probed with amouse monoclonal anti-SMN (1:5,000 BD Biosciences, San Jose, Calif.) anda rabbit polyclonal anti-β-tubulin (1:750, Santa Cruz Biotechnology,Santa Cruz, Calif.) antibodies. The membranes were incubated withinfrared secondary antibodies (1:20,000, LI-COR Biosciences, LincolnNB), and protein bands were visualized by quantitative fluorescenceusing Odyssey (LI-COR Biosciences). Molecular weight markers confirmedthe sizes of the bands.

Immunohistochemistry. For histological analysis, treated and untreatedmice were killed at 16 and 58-66 days were perfused with 4%paraformaldehyde (pH 7.4), the spinal cords were removed and placed in30% sucrose for 48-72 hours, embedded in OCT and cut into 10 μm frozensections by a cryostat. Spinal cord sections were blocked for 1 h atroom temperature (RT) and then incubated with either a mouse monoclonalanti-SMN antibody (1:200 dilution) to identify AAV-derived hSMNexpression, or a goat polyclonal anti-choline acetyl transferase (ChAT)antibody (Millipore; Burlington, Mass.; 1:100 dilution) to identifymotor neurons of laminae 8 and 9 (ventral horn) of the spinal cord or arabbit polyclonal anti-glial fibrillary acidic protein (GFAP) antibody(Sigma-Aldrich, 1:2,500 dilution) to detect astrocytes. Primaryantibodies were incubated for 1 h at RT followed by an overnightincubation at 4° C. in a humidified chamber. Spinal cord sections werethen incubated for 1 h at RT with either a FITC-conjugated anti-rabbitsecondary antibody or a Cy3-conjugated anti-goat secondary antibody(Jackson ImmunoResearch; West Grove, Pa.; 1:250 dilution). To increasethe SMN and ChAT immuno-positive signal, a TSA signal amplification kit(Perkin Elmer; Waltham, Mass.) or a citric acid antigen retrievalprotocol (Vector Labs; Burlingame, Calif.) were performed according tothe manufacturer's instruction, respectively. Sections werecover-slipped with Vectashield mounting media (Vector Labs; Burlingame,Calif.).

Motor neuron counting. The number of ChAT immuno-positive cells wascounted in the cervical, thoracic, and lumbar segments. Bilateral countswere performed at 100× magnification in the ventral horns along therostrocaudal axis of the three spinal cord segments. Adjacent sectionswere at least 100 microns apart to prevent double counting of the samecell. Special care was taken to compare anatomically matched sectionsbetween different animals, and all cell counts were assessed blind by asingle observer. Cells located in laminae 8 and 9 of the spinal cordexhibiting a fluorescent ChAT signal markedly above background wereconsidered motor neurons.

Myofiber size. For histological analysis of the periphery, the fixedquadriceps, gastrocnemius and intercostal muscles from the right side ofeach mouse were processed by paraffin and stained for hematoxylin-eosinto determine myofiber size as reported (Avila et al., J. Clin. Invest.(2007) 117:659-671). Approximately 500 non-overlapping myofibers fromeach muscle per animal were randomly selected and photographed at 60×magnification. The cross-section areas of each myofiber were measuredusing a Metamorph software (Molecular Devices, Sunnyvale, Calif.).

Neuromuscular Junction Staining (NMJ). The fixed muscle groups from theleft side of each mouse were stored in PBS for NMJ analysis. In totostaining on teased muscle fibers from the quadriceps, gastrocnemius andintercostals muscles was performed as reported (Lesbordes et al., Hum.Mol. Genet. (2003) 12:1233-1239). Pre-synaptic nerve terminals werelabeled by overnight incubation at 4° C. with a rabbit polyclonalantibody against the 150 kD neurofilament isoform (NF-M, Millipore,Billerica, Mass., 1:200 dilution), followed by a biotinylatedanti-rabbit secondary antibody (Jackson ImmunoResearch, 1:200 dilution).Acetylcholine receptors on the muscle endplates were labeled with Alexa555-conjugated α-bungarotoxin (Molecular Probes, Eugene, Oreg.) at1:5000 for 3 h at RT. Stained muscle fibers were mounted onto slides,cover-slipped with Vectashield, and viewed under epifluorescence. ForNMJ quantification, a minimum of 100 NMJs from each muscle per animalwere randomly selected and assessed under the microscope. Confocalimages were captured using a Zeiss LSM 510-META microscope.

Behavior tests. In the righting reflex, each mouse was placed on asupine position and the time taken for the mouse to reposition itselfonto all four paws was measured. The procedure was repeated three timesfor each animal, and the average of the three scores was designated therighting score. If the mouse did not respond within 60 seconds, the testwas terminated. In the negative geotaxis, each mouse was placed on a45°-platform facing downward. The test was deemed a success if the mouseturned 1800 to the “head up” position. Each mouse was given threeattempts to complete the task in 180 seconds or less. In the gripstrength, the forelimbs and hindlimbs were placed together on a wiregrid and gently dragging horizontally along the mesh. Resistance wasrecorded in grams by a force transducer. In the hindlimb splay test,each mouse was suspended by its tail for 5 seconds and the resultingsplay was scored based on an arbitrary system. A healthy splay of bothhindlimbs similar to that observed in wild-type mice was given a scoreof 4. An acute splay angle or “weak splay” of both hindlimbs was a scoreof 3. A single leg splay was assigned a score of 2. A mouse thatexhibited no splay was given a score of 1. Finally, a score of 0occurred when the pup pulled both hindlimbs together, effectivelycrossing them over the other.

Statistics. The behavioral tests, the number of motor neurons, thecross-section myofiber areas, and the NMJ were analyzed with one-wayANOVA and Bonferroni multiple post hoc comparisons and with unpairedtwo-tailed student t-tests. The Kaplan-Meier survival curve was analyzedwith the log-rank test equivalent to the Mantel-Haenszel test. Allstatistical analyses were performed with GraphPad Prism v4.0 (GraphPadSoftware, San Diego, Calif.). Values with p<0.05 were consideredsignificant.

Example 1 Significant Increase in Survival with Treatment UsingAAV-Mediated SMN1 Delivery

SMA mice on postnatal day 0 (P0) were injected intracerebroventricularlywith AAV-hSMN1 into both cerebral lateral ventricles and by directspinal cord injection into the upper lumbar spinal cord for a total doseof 5.0 e10 genome copies per mouse. Treated and untreated SMA mice wererandomly separated into either a survival cohort in which all the micewere left undisturbed and sacrificed at a humane end point, or into anage-matched cohort in which all the mice were sacrificed at 16 days forage-matched comparisons with end-stage untreated SMA mice.

In the survival cohort, SMA mice treated with AAV-hSMN1 showed asignificant increase in median lifespan to 50 days (p<0.0001), comparedto 15 days in untreated SMA controls (FIG. 1 ). All of the treated SMAmice were alive at 15 days, and 87.5% of the treated SMA mice were aliveat 19 days compared to 0% in untreated SMA. The Kaplan-Meier curveshowed a bimodal survival distribution with treatment, in which thefirst group died at 17-27 days and the second group at 58-66 days (FIG.1 ). In the first group, the majority of the treated SMA mice showedambulation, but the mice were stunted in growth and were ultimatelyfound dead in the cage. The second group of treated SMA mice at 58-66days showed ambulation and weight gain, but eventually developed severehindlimb necrosis that resulted in euthanasia of the animal. As such,the 58-66 days mice were analyzed in parallel with the 16-daysage-matched cohort.

Example 2 AAV-Mediated Expression of SMN in the Spinal Cord and MotorNeuron Counts

Levels of hSMN protein increased throughout the spinal cord followingCNS administration of AAV-hSMN1. In AAV-treated SMA mice at 16 days,there was an approximate 34.0- and 3.6-fold increase in hSMN proteinlevels in the injected lumbar segment compared to untreated SMA andwild-type mice, respectively (FIG. 2A). The increase in hSMN proteinexpression extended into the other segments, which included a >2.0-foldincrease above wild type levels in the thoracic and cervical spinal cordat 16 days (FIGS. 2B and 2C). In the second group, hSMN proteinexpression was sustained in AAV-treated SMA mice at 58-66 days. Theinjected lumbar and neighboring thoracic and cervical regions wasapproximately 2.5-, 2.2- and 1.2-fold higher than age-matched WTcontrols, respectively.

Immunostaining of tissue sections showed hSMN protein in the dorsal andventral horns of the spinal cord in treated SMA mice at 16 and 58-66days (FIG. 3 ). Upon closer examination of the transduced cells,vector-derived hSMN expression was detected in a punctate patternthroughout the cytosol, and in gem-like structures in the nucleus (FIG.3A). Furthermore, hSMN protein was localized to neurites in distinctgranule-like structures that could be seen spanning the length ofdendrites and axons (FIGS. 3B-3D). The very low level of endogenous hSMNprotein in SMA mice was below the threshold of immunodetection in cells(FIG. 3E).

Co-localization with ChAT and hSMN confirmed that a subset of thetransduced cells were indeed motor neurons (FIGS. 3F-3I). At 16 days,approximately 18-42% of ChAT-positive cells in the lumbar, thoracic andcervical segments of the spinal cord were transduced by AAV-hSMN1 (FIG.3J). This percentage was higher at 58-66 days, in which 60-70% of motorneurons expressed exogenous hSMN in the three spinal cord segments (FIG.3J). There was an overall increase in the number of motor neurons intreated SMA mice compared to untreated mutants (FIG. 4 ). However, therewere significantly less motor neurons in treated SMA mice compared towild type mice at 16 and 58-66 days (FIG. 4 ).

hSMN immunostaining of cervical tissue sections from untreated,Intracerebroventricular (ICV)-only injected, and lumbar-only injectedheterozygote mice was performed. ICV injections alone did not contributeto appreciable AAV transduction patterns in the brain but neverthelessgenerated substantial targeting of the cervical spinal cord that was notachievable with lumbar-only injections. In particular, the ICV-onlyinjections resulted in the cervical spinal cord expression of hSMN. Thiswas in contrast to intraparenchymal injection of the lumbar segment thatshowed very little transduction of the cervical spinal cord, presumablydue to the distal proximity from the injection site. On occasion, SMNimmunopositive signal that possessed a gem-like appearance was observedin the nucleus of untreated heterozygote and wild-type mice. However,this immunostaining pattern was not observed in the nucleus of untreatedSMA mice.

Thus, the combination of ICV and lumbar injections in P0 mice providedbroad, widespread transduction of the spinal cord. ICV injections ofAAV8-hSMN targeted the cervical spinal cord for transduction.

Example 3 Effects of AAV Treatment on Myofiber Size, the NMJ, andBehavior

The quadriceps (proximal), gastrocnemius (distal) and intercostal(respiratory) muscles were chosen for analysis because they show markeddegeneration. In untreated SMA mice at 16 days, myofibers were small andthe majority of individual cells contained a cross-section area of <100um² (FIG. 5A). Less than 10% of the myofibers from the untreated SMAmice contained a cross-section area of more than 200 um². In contrast,the distribution of myofiber sizes in AAV-hSMN1 treated SMA mice wassimilar to wild type, and many cells possessed a cross-section area ofmore than 200 and more than 400 μm² at 16 and 58-66 days, respectively(FIGS. 5A and 5B). The overall average at 16 days showed that themyofibers from treated SMA mice were more than 2-fold larger than thosefrom untreated SMA mice (FIG. 5C). Furthermore, the average myofibercross-section area in treated SMA mice at 58-66 days was 67%, 76%, and82% that of wild type mice in the quadriceps, gastrocnemius, andintercostal, respectively (FIG. 5C).

Analysis of the neuromuscular junction (NMJ) from untreated SMA mice at16 days showed abnormal accumulation of neurofilament protein at thepre-synaptic termini (FIG. 6A). Approximately 75-90% of the pre-synaptictermini from the quadriceps, gastrocnemius, and intercostal showed thishallmark pathology in untreated SMA mice (FIG. 6F). In contrast, themajority of the pre-synaptic termini from AAV-hSMN1 treated SMA mice didnot contain this collapsed structure (FIG. 6B, 6D). Only 10-25% and 5%of the pre-synaptic termini from treated SMA mice showed this hallmarkpathology at 16 and 58-66 days, respectively (FIG. 6F). However,treatment resulted in more branching at the pre-synaptic terminicompared to wild type (FIG. 6B-6E). On the post-synaptic NMJ fromtreated SMA and wild type mice, α-bungarotoxin staining produced a‘pretzel-like’ structure that was indicative of a functional network ofacetylcholine receptors (FIG. 6B-6E).

Treated and untreated mice were subjected to periodic behavioral teststhat have been validated for this animal model (Butchbach et al.,Neurobiol. Dis. (2007) 27:207-219; El-Khodar et al., Exp. Neurol. (2008)212:29-43). Treated SMA mice had good body scores and ambulatory skills,whereas untreated SMA mice were emancipated and paralyzed (FIG. 7A).Treated SMA mice were significantly heavier than untreated SMA controls,although they never reached wild-type size (FIG. 7B). Treated SMA miceshowed a significant improvement in righting latency (FIG. 7C). Therealso was a significant improvement in the treated SMA mice to completethe negative geotaxis test, which measures spatial locomotive behavior(FIG. 7D). Furthermore, treated SMA mice showed significant improvementsin grip strength and in the ability to splay their hindlimbs (FIG. 7E,7F).

Example 4 Effects on Longevity Using Self-Complementary AAV

Without being limited as to theory, self-complementary AAV (scAAV)vectors are predicted to have faster expression kinetics due to thedouble-stranded recombinant genome (reviewed in McCarty, D. M. Molec.Ther. (2008) 16:1648-1656). This rapid increase in expression may bebeneficial in highly aggressive diseases or conditions where thetemporal window of intervention is small. Thus, to determine whetherearlier expression could improve efficacy, a scAAV vector (scAAV-hSMN1)was engineered and tested. Using the same site of injections asperformed in Example 1, a dose of 1.7 e10 genome copies of scAAV-hSMN1was administered into P0 SMA mice.

Treatment with scAAV-hSMN1 resulted in a striking and remarkableimprovement in median survival of 157 days (p<0.0001), which was a +214%and +881% increase compared to AAV-hSMN1-treated and untreated SMA mice,respectively (FIG. 8 ). Approximately 42% of the scAAV-treated micepossessed a more than 1000% increase (log-fold increase) in mediansurvival. Furthermore, scAAV2/8-GUSB-hSMN1 treatment resulted in 88% ofthe SMA mice living beyond 66 days, in contrast to 0% withAAV2/8-CBA-hSMN1. The scAAV-treated SMA mice possessed healthy bodyscores, were well groomed, gained weight, and maintained ambulationthroughout their life. Interestingly, scAAV-treated SMA mice developedonly mild hindlimb necrosis that never progressed into a severephenotype. The majority of scAAV-treated SMA mice were sacrificed due toan unforeseen and sudden appearance of respiratory distress, whichincluded audible clicking gasps when breathing and a decreased rate ofrespiration.

To better understand the basis for the observed increase in survivalwith scAAV8-hSMN, additional SMA mice were treated at P0 and sacrificedat 16 or 64 (58-66d) days post-injection, and analyzed with thelong-lived scAAV8-treated mice from the survival curve (FIG. 8 ). At 16days, SMN expression levels from the scAAV8-hSMN group wereapproximately 60-90% to those observed in WT animals. These levels weresubstantially less than that achieved with AAV8-hSMN treatment at thistime point. In the scAAV8-hSMN-treated SMA mice, SMN levels in both thelumbar and thoracic segments were above or at WT levels at 58-66 and120-220 days, respectively (FIGS. 2A and 2B). In contrast, SMN levels inthe cervical spinal cord remained relatively low at all time points.

Comparison of AAV vector tropism in the lumbar spinal cord was examinedusing hSMN immunostaining on frozen tissue sections from untreated SMA,AAV8-hSMN-treated SMA, and scAAV8-hSMN-treated SMA mice at 16 days and157 days post-injection. A diffuse hSMN immunostaining patternconsistent with glial cell morphology was observed at 16 days withAAV8-hSMN. Doubling immunolabeling of hSMN and mGFAP confirmed that asubset of the AAV8-hSMN-transduced cells were astrocytes. In contrast,scAAV8 treatment resulted in hSMN expression only in distinct cellbodies with neuronal morphology, which did not co-localize with GFAP.Double immunolabeling of hSMN and the motor neuron marker mChATconfirmed that a subset of cells transduced by scAAV8-hSMN and AAV8-hSMNwere motor neurons. hSMN expression was also observed in theinterneuronal cell layers of the spinal cord with both viral vectors, asexemplified by scAAV8-hSMN at 157 days. Thus, in contrast to AAV8-hSMN,histological analysis of scAAV8-hSMN-treated SMA mice showed hSMNexpression was largely restricted to neurons.

Furthermore, double immunostaining with hSMN and mChAT showed asignificant increase in the percentage of motor neurons transduced withscAAV8-hSMN compared to AAV8-hSMN (FIG. 10A). The more efficienttargeting of motor neurons with scAAV correlated with a significantincrease in the number of ChAT-positive cells (FIGS. 10B-10D). Analysisof the NMJ in the quadriceps and intercostal muscles at 16 days alsoshowed a significant decrease in the number of collapsed structures withscAAV-hSMN compared to AAV8-hSMN (FIGS. 10E and 10F). However, there wasan increase in the number of aberrant NMJs at 216-269 days that wasconcomitant with the decline of motor neuron cell counts in thescAAV-hSMN group (FIGS. 10B-10F).

To summarize, injection of AAV8-hSMN at birth into the CNS of a mousemodel of SMA resulted in widespread expression of SMN throughout thespinal cord that translated to a robust improvement in skeletal musclephysiology. Treated SMA animals also displayed significant improvementson behavioral tests indicating that the neuromuscular junction wasfunctional. Importantly, treatment with AAV8-hSMN increased the medianlifespan of SMA mice to 50 days compared to 15 days for untreatedcontrols. Moreover, SMA mice injected with a self-complementary AAVvector resulted in improved efficacy including a significant extensionin median survival to 157 days. These data evidence that CNS-directed,AAV-mediated SMN augmentation is highly efficacious in addressing boththe neuronal and muscular pathologies of a severe mouse model of SMA.

Thus, compositions and methods for treating spinal cord disorders aredisclosed. Although preferred embodiments of the subject invention havebeen described in some detail, it is understood that obvious variationscan be made without departing from the spirit and the scope of theinvention as defined herein.

1-19. (canceled)
 20. A method of treating spinal muscular atrophy (SMA)comprising administering to a subject in need thereof a therapeuticallyeffective amount of a recombinant vector comprising a heterologousnucleic acid construct, wherein the heterologous nucleic acid constructcomprises: a. a first AAV2 inverted terminal repeat (ITR); b.cytomegalovirus enhancer/chicken-β actin (CBA) promoter; c. apolynucleotide encoding a survival motor neuron (SMN) protein comprisingthe amino acid sequence of SEQ ID NO: 2; and d. a second AAV2 ITR. 21.The method of claim 20, wherein the second ITR is mutated.
 22. Themethod of claim 21, wherein the second ITR comprises a mutation in aterminal resolution sequence.
 23. The method of claim 20, wherein theheterologous nucleic acid construct is a self-complementary recombinantAAV (scAAV).
 24. The method of claim 20, wherein the recombinant vectoris a viral vector.
 25. The method of claim 24, wherein the viral vectoris a recombinant AAV (rAAV) virion.
 26. The method of claim 25, whereinabout 10⁶ to about 10¹⁵ rAAV virions are administered.
 27. The method ofclaim 20, wherein the rAAV virions are administered in a single-dose.28. A method of treating spinal muscular atrophy (SMA) comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a recombinant vector comprising a heterologous nucleic acidconstruct, wherein the heterologous nucleic acid construct comprises: a.a first AAV2 inverted terminal repeat (ITR); b. cytomegalovirusenhancer/chicken-β actin (CBA) promoter; c. a polynucleotide encoding asurvival motor neuron (SMN) protein comprising the amino acid sequencethat is 90% identical to SEQ ID NO: 2; and d. a second AAV2 ITR.
 29. Themethod of claim 28, wherein the polynucleotide encoding the SMN proteincomprises an amino acid sequence that is 95% identical to SEQ ID NO: 2.30. The method of claim 28, wherein the second ITR is mutated.
 31. Themethod of claim 30, wherein the second ITR comprises a mutation in aterminal resolution sequence.
 32. The method of claim 28, wherein theheterologous nucleic acid construct is a self-complementary recombinantAAV (scAAV).
 33. The method of claim 28, wherein the recombinant vectoris a viral vector.
 34. The method of claim 33, wherein the viral vectoris a recombinant AAV (rAAV) virion.
 35. The method of claim 34, whereinabout 10⁶ to about 10¹⁵ rAAV virions are administered.
 36. The method ofclaim 28, wherein the rAAV virions are administered in a single-dose.37. A method of treating spinal muscular atrophy (SMA) comprisingadministering to a subject in need thereof about 10⁶ to about 10¹⁵recombinant AAV (rAAV) virions, wherein the rAAV virions comprise aself-complementary heterologous nucleic acid construct comprising: a. afirst AAV2 inverted terminal repeat (ITR); b. cytomegalovirusenhancer/chicken-β actin (CBA) promoter; c. a polynucleotide encoding asurvival motor neuron (SMN) protein comprising the amino acid sequencethat is at least 95% identical to SEQ ID NO: 2; and d. a second AAV2ITR, wherein the second ITR comprises a mutation in a terminalresolution sequence.
 38. The method of claim 37, wherein thepolynucleotide encoding the SMN protein comprises an amino acid sequencethat is identical to SEQ ID NO:
 2. 39. The method according to claim 37,wherein the rAAV virions are administered in a single-dose.
 40. A methodof treating spinal muscular atrophy (SMA) comprising administering to asubject in need thereof a therapeutically effective amount ofrecombinant AAV (rAAV) virions, wherein the rAAV virions comprise aself-complementary heterologous nucleic acid construct comprising, in 5′to 3′ order, a wild-type AAV2 ITR; a cytomegalovirus enhancer/chicken-βactin (CBA) promoter; a polynucleotide encoding a survival motor neuron(SMN) protein comprising an amino acid sequence that is at least 95%identical to SEQ ID NO: 2; a mutated AAV2 ITR comprising a mutation in aterminal resolution sequence; an inverted polynucleotide encoding asurvival motor neuron (SMN) protein comprising an amino acid sequencethat is at least 95% identical to SEQ ID NO: 2; an invertedcytomegalovirus enhancer/chicken-β actin (CBA) promoter; and an invertedwild-type AAV2 ITR.
 41. The method of claim 40, wherein thepolynucleotide encoding the SMN protein comprises an amino acid sequencethat is identical to SEQ ID NO:
 2. 42. The method of claim 40, whereinthe inverted polynucleotide encoding the SMN protein comprises an aminoacid sequence that is identical to SEQ ID NO:
 2. 43. The method of claim40, wherein the polynucleotide encoding the SMN protein comprises anamino acid sequence that is identical to SEQ ID NO: 2, and wherein theinverted polynucleotide encoding the SMN protein comprises an amino acidsequence that is identical to SEQ ID NO:
 2. 44. The method according toclaim 40, wherein the rAAV virions are administered in a single-dose.45. The method according to claim 40, wherein about 10⁶ to about 10¹⁵rAAV virions are administered.
 46. The method of claim 40, wherein therAAV virions are administered in a single-dose, and wherein about 10⁶ toabout 10¹⁵ rAAV virions are administered.